Multiple-variable dose regimen for treating idiopathic inflammatory bowel disease

ABSTRACT

Multiple-variable dose methods for treating TNFα-related disorders, including Crohn&#39;s disease and psoriasis, comprising administering TNFα inhibitors, including TNFα antibodies, are described. Multiple-variable dose methods include administration of a TNF-inhibitor in an induction or loading phase followed by administration of the agent in a maintenance or treatment phase, wherein the TNF-inhibitor is administered in a higher dosage during the induction phase.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/229,664 (now allowed), filed Mar. 28, 2014, which is a continuationof U.S. patent application Ser. No. 11/104,117, filed Apr. 11, 2005, nowU.S. Pat. No. 8,889,136, which claims the benefit of U.S. ProvisionalApplication No. 60/561,139, filed Apr. 9, 2004; U.S. ProvisionalApplication No. 60/561,710, filed Apr. 12, 2004; and U.S. ProvisionalApplication No. 60/569,100, filed May 7, 2004, the contents of each ofwhich applications are hereby incorporated by reference in theirentirety.

This application is related to U.S. Pat. Nos. 6,090,382, 6,258,562, and6,509,015. This application is also related to U.S. patent applicationSer. No. 09/801,185, filed Mar. 7, 2001; U.S. patent application Ser.No. 10/302,356, filed Nov. 22, 2002; U.S. patent application Ser. No.10/163,657, filed Jun. 5, 2002; U.S. patent application Ser. No.10/133,715, filed Apr. 26, 2002; U.S. patent application Ser. No.10/222,140, filed Aug. 16, 2002; U.S. patent application Ser. No.10/693,233, filed Oct. 24, 2003; U.S. patent application Ser. No.10/622,932, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,039, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,076, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,065, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,928, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,075, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,035, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,683, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,205, filed Jul. 18, 2003; U.S. patent application Ser. No.10/622,210, filed Jul. 18, 2003; U.S. patent application Ser. No.10/623,318, filed Jul. 18, 2003; and U.S. patent application Ser. No.10/422,287, filed Apr. 24, 2003. The entire contents of each of thesepatents and patent applications are hereby incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically as a text file in ASCII format and is herebyincorporated by reference in its entirety. Said text file, created onFeb. 20, 2015, is named 110222-0010-110_SL.txt and is 12,521 bytes insize.

BACKGROUND OF THE INVENTION

Cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)are molecules produced by a variety of cells, such as monocytes andmacrophages, which have been identified as mediators of inflammatoryprocesses. Cytokines, including TNF, regulate the intensity and durationof the inflammatory response which occurs as the result of an injury orinfection. Elevated levels of TNF play an important role in pathologicinflammation. TNF also referred to as (TNFα) has been implicated in thepathophysiology of a variety of human diseases and disorders, includingsepsis, infections, autoimmune diseases, transplant rejection andgraft-versus-host disease (see e.g., Moeller et al. (1990) Cytokine2:162; U.S. Pat. No. 5,231,024 to Moeller et al.; European PatentPublication No. 260 610 B1 by Moeller, A. et al.; Vasilli (1992) Annu.Rev. Immunol. 10:411; Tracey and Cerami (1994) Annu. Rev. Med. 45:491).

TNF has been implicated in psoriasis. Expression of TNF-induced proteinsand the presence of activated T lymphocytes in psoriatic plaques but notuninvolved skin, suggest their involvement in the pathogenesis of thedisease. There are several types of psoriasis according to cutaneousmanifestations: plaque psoriasis, guttate psoriasis, erythrodermicpsoriasis, generalized pustular and localized pustular psoriasis. Plaquepsoriasis is the most common type, however. Treatment of psoriasisdepends on the extent of the disease. Topical corticosteroids arecommonly used for mild to moderate localized cases. Keratolytic agentsand coal tar are also used as topical medications, and phototherapy iscommonly used for more widespread disease. Other systemic therapy, suchas methotrexate cyclosporine and synthetic retinoids are effective, butare often administered in rotation due to their possible cumulativetoxic effect.

TNF has also been implicated in Crohn's disease. Crohn's is diagnosed onthe basis of clinical, endoscopic, radiographic, and histologiccriteria. The treatment of Crohn's disease is challenging. Treatment isbased on location, extent, and severity of disease. Current compoundsand regimens do not completely abate the inflammatory process and havesignificant side effects.

SUMMARY OF THE INVENTION

There is a need to treat TNFα-related disorders, where TNFα activity isdetrimental, in a safe and effective manner. The present inventionincludes multiple-variable dose methods for improved treatment ofTNFα-related disorders where TNFα activity is detrimental.

The invention describes a multiple-variable dose method for treating adisorder in which TNFα activity is detrimental, comprising administeringto a subject in need thereof at least one induction dose of a TNFαinhibitor such that a threshold level of TNFα inhibitor is achievedwithin an induction phase; and subsequently administering to the subjectat least one treatment dose of the TNFα inhibitor within a treatmentphase, such that treatment occurs.

The invention also describes a multiple-variable dose method fortreating Crohn's disease, comprising administering to a subject in needthereof at least one induction dose of a TNFα inhibitor such that athreshold level of TNFα inhibitor is achieved within an induction phase;and subsequently administering to the subject at least one treatmentdose of the TNFα inhibitor within a treatment phase, such that treatmentoccurs. The multiple-variable dose method of the invention can also beused to treat ulcerative colitis or psoriasis. In another embodiment,multiple-variable dose method of the invention is used to treat aspsoriasis in combination with psoriatic arthritis.

The invention includes a multiple-variable dose method of inducingremission of Crohn's disease, comprising administering to a subject inneed thereof at least one induction dose of a TNFα inhibitor such that athreshold level of TNFα inhibitor is achieved within an induction phase;and subsequently administering to the subject at least one treatmentdose of the TNFα inhibitor within a treatment phase, such that treatmentoccurs.

In an additional embodiment, the invention includes a multiple-variabledose method of reducing psoriatic plaques comprising administering to asubject in need thereof at least one induction dose of a TNFα inhibitorsuch that a threshold level of TNFα inhibitor is achieved within aninduction phase; and subsequently administering to the subject at leastone treatment dose of the TNFα inhibitor within a treatment phase, suchthat treatment occurs.

In one embodiment, the TNFα inhibitor is etanercept or infliximab.

In one embodiment of the invention, the TNFα inhibitor is a TNFαantibody, or antigen-binding fragment thereof. In another embodiment ofthe invention, the TNFα inhibitor is a human TNFα antibody, orantigen-binding fragment thereof. In one embodiment, the antibody is anisolated human antibody, or an antigen-binding portion thereof, thatdissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less. In anotherembodiment, the antibody has the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In still another embodiment, the antibody has a light chain variableregion (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and aheavy chain variable region (HCVR) comprising the amino acid sequence ofSEQ ID NO:2. In a further embodiment, the antibody is D2E7.

The methods of the invention can be used to treat a TNFα-relateddisorder selected from the group consisting of an autoimmune disease, aninfectious disease, transplant rejection or graft-versus-host disease,malignancy, a pulmonary disorder, an intestinal disorder, a cardiacdisorder, sepsis, a spondyloarthropathy, a metabolic disorder, anemia,pain, a hepatic disorder, a skin disorder, a nail disorder, andvasculitis. In one embodiment, the autoimmune disorder is selected fromthe group consisting of rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, gouty arthritis, allergy, multiple sclerosis, autoimmunediabetes, autoimmune uveitis, and nephrotic syndrome. In anotherembodiment, the TNFα-related disorder is selected from the groupconsisting of inflammatory bone disorders, bone resorption disease,alcoholic hepatitis, viral hepatitis, fulminant hepatitis, coagulationdisturbances, burns, reperfusion injury, keloid formation, scar tissueformation, pyrexia, periodontal disease, obesity, and radiationtoxicity. In still another embodiment, the TNFα-related disorder isselected from the group consisting of Behcet's disease, ankylosingspondylitis, asthma, chronic obstructive pulmonary disorder (COPD),idiopathic pulmonary fibrosis (IPF), restenosis, diabetes, anemia, pain,a Crohn's disease-related disorder, juvenile rheumatoid arthritis (JRA),a hepatitis C virus infection, psoriatic arthritis, and chronic plaquepsoriasis.

In one embodiment of the invention, the TNFα-related disorder is Crohn'sdisease. In another embodiment, the disorder is ulcerative colitis. Instill another embodiment, the disorder is psoriasis. In still anotherembodiment, the disorder is psoriasis in combination with psoriaticarthritis (PsA). In still another embodiment, the TNFα-related disorderis rheumatoid arthritis.

In one embodiment, the treatment dose is 40-60% of the induction dose.

In one embodiment, the induction dose used in the multiple variable doseregimen of the invention ranges from about 20 to 200 mg. In anotherembodiment, the induction dose ranges from about 80 to 160 mg.

In one embodiment, the treatment dose used in the multiple variable doseregimen of the invention ranges from about 20 to 120 mg. In anotherembodiment, the treatment dose ranges from about 40 to 80 mg.

In one embodiment of the invention, the induction dose comprises about160 mg. In another embodiment, the treatment dose comprises about 80 mg.

In one embodiment of the invention, the induction dose comprises about80 mg. In still another embodiment, the treatment dose comprises about40 mg.

In one embodiment, the induction dose used in the multiple variable doseregimen of the invention ranges from 20 to 200 mg. In anotherembodiment, the induction dose ranges from 80 to 160 mg. In oneembodiment, the treatment dose used in the multiple variable doseregimen of the invention ranges from 20 to 120 mg. In anotherembodiment, the treatment dose ranges from 40 to 80 mg.

In still another embodiment of the invention, the induction dosecomprises 160 mg. In yet another embodiment, the treatment dosecomprises 80 mg.

In one embodiment of the invention, the induction dose comprises 80 mg.

In yet another embodiment, the treatment dose comprises 40 mg.

In one embodiment, the treatment dose is administered about 2 weeksfollowing the induction dose.

In one embodiment, the TNFα inhibitor is administered subcutaneously. Inanother embodiment, the TNFα inhibitor is administered in combinationwith methotrexate. The methotrexate can be administered, for example, ina dose of between 2.5 mg and 30 mg.

In one embodiment, the threshold level of a multiple dose method oftreatment of Crohn's disease is determined by a reduction in thesubject's Crohn's Disease Activity Index (CDAI) score.

In one embodiment, the threshold level of a multiple dose method oftreatment of psoriasis is determined as a therapeutic effect selectedfrom the group consisting of a reduction in psoriatic plaques, animprovement in the subject's Psoriatic Area Severity Index (PAST), andan improvement in the subject's Physician's Global Assessment (PGA)score.

The invention describes a multiple-variable dose method of inducingremission of Crohn's disease, comprising administering to a subject inneed thereof at least one induction dose of D2E7 such that a thresholdlevel of TNFα inhibitor is achieved within an induction phase; andsubsequently administering to the subject at least one treatment dose ofD2E7 within a treatment phase, such that treatment occurs.

In another embodiment, the invention includes a multiple-variable dosemethod of reducing psoriatic plaques comprising: administering to asubject in need thereof at least one induction dose of D2E7 such that athreshold level of TNFα inhibitor is achieved within an induction phase;and subsequently administering to the subject at least one treatmentdose of the D2E7 within a treatment phase, such that treatment occurs.

The invention provides a kit for the treatment of a disorder in whichTNFα activity is detrimental comprising:

-   -   a) at least one container comprising an induction dose of a TNFα        inhibitor;    -   b) at least one container comprising a treatment dose a TNFα        inhibitor; and    -   c) instructions for administration of the induction dose within        an induction phase and the treatment dose of the TNFα inhibitor        within a treatment phase.

The invention also describes a kit for the treatment of a disorder inwhich TNFα activity is detrimental, comprising at least one containercomprising an induction dose of a TNFα inhibitor packaged withinstructions for administration of the induction dose within aninduction phase.

The invention describes a kit for the treatment of a disorder in whichTNFα activity is detrimental, comprising at least one containercomprising a treatment dose of a TNFα inhibitor packaged withinstructions for administration of the treatment dose within a treatmentphase.

In one embodiment of the invention, the kit is used for the treatmentdisorder is selected from the group consisting of an autoimmune disease,an infectious disease, transplant rejection or graft-versus-hostdisease, malignancy, a pulmonary disorder, an intestinal disorder, acardiac disorder, sepsis, a spondyloarthropathy, a metabolic disorder,anemia, pain, a hepatic disorder, a skin disorder, a nail disorder, andvasculitis. In one embodiment, the autoimmune disorder is selected fromthe group consisting of rheumatoid arthritis, rheumatoid spondylitis,osteoarthritis, gouty arthritis, allergy, multiple sclerosis, autoimmunediabetes, autoimmune uveitis, and nephrotic syndrome. In still anotherembodiment, the TNFα-related disorder is selected from the groupconsisting of Behcet's disease, ankylosing spondylitis, asthma, chronicobstructive pulmonary disorder (COPD), idiopathic pulmonary fibrosis(IPF), restenosis, diabetes, anemia, pain, a Crohn's disease-relateddisorder, juvenile rheumatoid arthritis (JRA), a hepatitis C virusinfection, psoriatic arthritis, and chronic plaque psoriasis.

In one embodiment of the invention, the kit is used for the treatmentdisorder is selected from the group consisting of Crohn's disease,ulcerative colitis, psoriasis in combination with psoriatic arthritis,and psoriasis.

In another embodiment, the TNFα inhibitor in the kit is a TNFα antibody,or antigen-binding fragment thereof. In one embodiment, the antibody isan isolated human antibody, or an antigen-binding portion thereof, thatdissociates from human TNFα with a K_(d) of 1×10⁻⁸ M or less and aK_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determined by surfaceplasmon resonance, and neutralizes human TNFα cytotoxicity in a standardin vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less. In anotherembodiment, the antibody has the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

In still another embodiment, the antibody has a light chain variableregion (LCVR) comprising the amino acid sequence of SEQ ID NO:1 and aheavy chain variable region (HCVR) comprising the amino acid sequence ofSEQ ID NO:2. In yet another embodiment, the antibody is D2E7.

In one embodiment, the TNFα inhibitor of the kit of the invention isetanercept or infliximab.

In one embodiment, the treatment dose provided in the kit is 40-60% ofthe induction dose.

In one embodiment, the induction dose provided in the kit ranges fromabout 20 to 200 mg. In another embodiment, the induction dose providedin the kit ranges from 80 to 160 mg.

In one embodiment, the treatment dose provided in the kit ranges fromabout 20 to 120 mg. In another embodiment, the treatment dose providedin the kit ranges from about 40 to 80 mg.

In one embodiment, the induction dose provided in the kit of theinvention comprises about 160 mg. In another embodiment, the treatmentdose comprises about 80 mg. In still another embodiment, the inductiondose comprises about 80 mg. In yet another embodiment, the treatmentdose comprises about 40 mg.

In one embodiment, the induction dose provided in the kit of theinvention comprises 160 mg. In another embodiment, the treatment dosecomprises 80 mg. In still another embodiment, the induction dosecomprises 80 mg. In yet another embodiment, the treatment dose comprises40 mg.

In yet another embodiment, the container is a pre-filled syringe. Instill another embodiment, the kit contains instructions foradministering the treatment dose 2 weeks following the induction dose.

The invention also provides method for treating a disorder in which TNFαactivity is detrimental, comprising administering to a subject in needthereof, a single dose of a TNFα inhibitor such that the disorder istreated. In one embodiment, the TNFα inhibitor is an anti-TNFα antibody,or an antigen binding portion thereof. In another embodiment, the TNFαinhibitor is a human anti-TNFα antibody, or antigen binding portionthereof, including, for example, a human antibody, or an antigen-bindingportion thereof, dissociates from human TNFα with a K_(d) of 1×10⁻⁸ M orless and a K_(off) rate constant of 1×10⁻³ s⁻¹ or less, both determinedby surface plasmon resonance, and neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁷ M or less. In oneembodiment, said human antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) rate constant of 5×10⁻⁴ s⁻¹or less. In another embodiment, said human antibody, or antigen-bindingportion thereof, dissociates from human TNFα with a K_(off) rateconstant of 1×10⁻⁴ s⁻¹ or less. In still another embodiment, said humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less. In yet another embodiment, said human antibody, orantigen-binding portion thereof, neutralizes human TNFα cytotoxicity ina standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁹ M or less. In afurther embodiment, said human antibody, or antigen-binding portionthereof, neutralizes human TNFα cytotoxicity in a standard in vitro L929assay with an IC₅₀ of 1×10⁻¹⁰ M or less. The human antibody, orantigen-binding portion thereof, can also be a recombinant antibody, orrecombinant antigen-binding portion thereof. In one embodiment, saidhuman antibody, or an antigen-binding portion thereof, is D2E7. Inanother embodiment, the single dose is selected from the groupconsisting of about 80 mg, 40, mg, and 20 mg. In still anotherembodiment, the administration is by subcutaneous injection. In oneembodiment of the invention, the TNFα-related disorder is Crohn'sdisease. In another embodiment, the disorder is ulcerative colitis. Instill another embodiment, the disorder is psoriasis. In still anotherembodiment, the disorder is psoriasis in combination with psoriaticarthritis (PsA). In still another embodiment, the TNFα-related disorderis rheumatoid arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides results demonstrating the percentage of patient's withCrohn's disease remission (CDAI<150) over time using themultiple-variable dose regimen.

FIG. 2 shows a graph of the decrease in the mean CDAI score of Crohn'spatients receiving the multiple variable dose regimens over time.

FIG. 3 (shown as FIG. 3A and FIG. 3B) shows remission and clinicalresponse in Crohn's patients receiving multiple variable dose treatment.FIG. 3A graphically provides the percentage of patients with a ≧70 pointCDAI decrease at four weeks. The P-values represent comparison with theplacebo group. FIG. 3B provides a graph of the percentage of patientswith a CDAI decrease ≧70 over time; *p=0.015 vs. placebo and **p=0.008vs. placebo.

FIG. 4 (shown as FIG. 4A and FIG. 4B) shows remission and clinicalresponse in Crohn's patients receiving multiple variable dose treatment.FIG. 4A graphically provides the percentage of patients with a ≧100point CDAI decrease at four weeks (p-values represent comparison withthe placebo group). FIG. 4B shows the percentage of patients with a CDAIdecrease ≧100 over time; *p=0.002 vs. placebo.

FIG. 5 provides results showing the median CRP levels in Crohn'spatients receiving multiple-variable treatments versus the placebo.

FIG. 6 provides results of the efficacy of the multiple-variable doseregimen at treating Crohn's disease as measured by the IBDQ score atfour weeks (P-values represent comparison with placebo group).

FIG. 7 shows the percentage of psoriasis patients with ≧PASI 50/75/90response at week 12 following treatment with each multiple variable D2E7dose and the placebo.

FIG. 8 provides results of the mean percentage PASI (Psoriasis Area andSeverity Index) improvement over a 12 week treatment (eow=every otherweek; *=p<0.001 vs. placebo).

FIG. 9 shows a comparative graph of the efficacy response at week 12 andweek 24 for patients with psoriasis and PsA.

FIG. 10 shows a comparative graph of the efficacy response at week 12and week 24 for patients with psoriasis and without PsA.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined.

The term “human TNFα” (abbreviated herein as hTNFα, or simply hTNF), asused herein, is intended to refer to a human cytokine that exists as a17 kD secreted form and a 26 kD membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kD molecules. The structure of hTNFα is describedfurther in, for example, Pennica, D., et al. (1984) Nature 312:724-729;Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y.,et al. (1989) Nature 338:225-228. The term human TNFα is intended toinclude recombinant human TNFα (rhTNFα), which can be prepared bystandard recombinant expression methods or purchased commercially (R & DSystems, Catalog No. 210-TA, Minneapolis, Minn.). TNFα is also referredto as TNF.

The term “TNFα inhibitor” includes agents which interfere with TNFαactivity. Examples of TNFα inhibitors include etanercept (Enbrel®,Amgen), infliximab (Remicade®, Johnson and Johnson), human anti-TNFmonoclonal antibody (D2E7/HUMIRA®, Abbott Laboratories), CDP 571(Celltech), and CDP 870 (Celltech), as well as other compounds whichinhibit TNFα activity, such that when administered to a subjectsuffering from or at risk of suffering from a disorder in which TNFαactivity is detrimental, the disorder is treated. The term also includeseach of the anti-TNFα human antibodies and antibody portions describedherein as well as those described in U.S. Pat. Nos. 6,090,382;6,258,562; 6,509,015, and in U.S. patent application Ser. Nos.09/801,185 and 10/302,356, each incorporated by reference herein.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavy(H) chains and two light (L) chains inter-connected by disulfide bonds.Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as HCVR or VH) and a heavy chain constant region.The heavy chain constant region is comprised of three domains, CHL CH2and CH3. Each light chain is comprised of a light chain variable region(abbreviated herein as LCVR or VL) and a light chain constant region.The light chain constant region is comprised of one domain, CL. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each VH and VL is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the inventionare described in further detail in U.S. Pat. Nos. 6,090,382; 6,258,562;and 6,509,015, and in U.S. patent application Ser. Nos. 09/801,185 and10/302,356, each of which is incorporated herein by reference in itsentirety.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Theantibody portions of the invention are described in further detail inU.S. Pat. Nos. 6,090,382, 6,258,562, 6,509,015, and in U.S. patentapplication Ser. Nos. 09/801,185 and 10/302,356, each of which isincorporated herein by reference in its entirety.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

A “conservative amino acid substitution”, as used herein, is one inwhich one amino acid residue is replaced with another amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art, including basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell (describedfurther below), antibodies isolated from a recombinant, combinatorialhuman antibody library (described further below), antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic for humanimmunoglobulin genes (see e.g., Taylor, L. D. et al. (1992) Nucl. AcidsRes. 20:6287) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies are subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hTNFα is substantially free of antibodies that specifically_(—)bind antigens other than hTNFα). An isolated antibody that specificallybinds hTNFα may, however, have cross-reactivity to other antigens, suchas TNFα molecules from other species (discussed in further detailbelow). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

A “neutralizing antibody”, as used herein (or an “antibody thatneutralized hTNFα activity”), is intended to refer to an antibody whosebinding to hTNFα results in inhibition of the biological activity ofhTNFα. This inhibition of the biological activity of hTNFα can beassessed by measuring one or more indicators of hTNFα biologicalactivity, such as hTNFα-induced cytotoxicity (either in vitro or invivo), hTNFα-induced cellular activation and hTNFα binding to hTNFαreceptors. These indicators of hTNFα biological activity can be assessedby one or more of several standard in vitro or in vivo assays known inthe art (see U.S. Pat. No. 6,090,382). Preferably, the ability of anantibody to neutralize hTNFα activity is assessed by inhibition ofhTNFα-induced cytotoxicity of L929 cells. As an additional oralternative parameter of hTNFα activity, the ability of an antibody toinhibit hTNFα-induced expression of ELAM-1 on HUVEC, as a measure ofhTNFα-induced cellular activation, can be assessed.

The term “surface plasmon resonance”, as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Example 1 of U.S. Pat. No. 6,258,562 andJönsson et al. (1993) Ann. Biol. Clin. 51:19; Jonsson et al. (1991)Biotechniques 11:620-627; Johnsson et al. (1995) J. Mol. Recognit.8:125; and Johnnson et al. (1991) Anal. Biochem. 198:268.

The term “K_(off)”, as used herein, is intended to refer to the off rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

The term “IC₅₀” as used herein, is intended to refer to theconcentration of the inhibitor required to inhibit the biologicalendpoint of interest, e.g., neutralize cytotoxicity activity.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind hTNFα, is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than hTNFα, which othersequences may naturally flank the nucleic acid in human genomic DNA.Thus, for example, an isolated nucleic acid of the invention encoding aVH region of an anti-hTNFα antibody contains no other sequences encodingother VH regions that bind antigens other than hTNFα.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein.

The term “dose,” as used herein, refers to an amount of TNFα inhibitorwhich is administered to a subject.

The term “multiple-variable dose” includes different doses of a TNFαinhibitor which are administered to a subject for therapeutic treatment.“Multiple-variable dose regimen” or “multiple-variable dose therapy”describe a treatment schedule which is based on administering differentamounts of TNFα inhibitor at various time points throughout the courseof treatment. In one embodiment, the invention describes amultiple-variable dose method of treatment comprising an induction phaseand a treatment phase, wherein a TNFα inhibitor is administered at ahigher dose during the induction phase than the treatment phase.

The term “induction phase” or “loading phase”, as used herein, refers toa period of treatment comprising administration of a TNFα inhibitor to asubject in order to attain a threshold level. During the inductionphase, at least one induction dose of TNFα inhibitor is administered toa subject suffering from a disorder in which TNFα is detrimental.

The term “threshold level”, as used herein, refers to a therapeuticallyeffective level of a TNFα inhibitor in a subject. A threshold level isachieved by administering at least one induction dose during theinduction phase of treatment. Any number of induction doses may beadministered to achieve a threshold level of TNFα inhibitor. Once athreshold level is achieved, the treatment phase is initiated.

The term “induction dose” or “loading dose,” used interchangeablyherein, refers to the first dose of TNFα inhibitor, which is larger incomparison to the maintenance or treatment dose. The induction dose canbe a single dose or, alternatively, a set of doses. The induction doseis often used to bring the drug in the body to a steady state amount,and may be used to which to achieve maintenance drug levels quickly. Aninduction dose is subsequently followed by administration of smallerdoses of TNFα inhibitor, i.e., the treatment dose. The induction dose isadministered during the induction phase of therapy. In one embodiment ofthe invention, the induction dose is at least twice the given amount ofthe treatment dose. In another embodiment of the invention, theinduction dose of D2E7 is 160 mg. In another embodiment, the inductiondose of D2E7 is 80 mg.

The term “treatment phase” or “maintenance phase”, as used herein,refers to a period of treatment comprising administration of a TNFαinhibitor to a subject in order to maintain a desired therapeuticeffect. The treatment phase follows the induction phase, and, therefore,is initiated once a threshold level is achieved.

The term “treatment dose” or “maintenance dose” is the amount of TNFαinhibit or taken by a subject to maintain or continue a desiredtherapeutic effect. A treatment dose is administered subsequent to theinduction dose. A treatment dose can be a single dose or, alternatively,a set of doses. A treatment dose is administered during the treatmentphase of therapy. Treatment doses are smaller than the induction doseand can be equal to each other when administered in succession. In oneembodiment, the invention describes at least one induction dose of D2E7of about 160 mg, followed by at least one treatment dose of about 80 mg.In another embodiment, the invention describes at least one inductiondose of D2E7 of about 80 mg, followed by at least one treatment dose ofabout 40 mg. In still another embodiment, the treatment dose isadministered at least two weeks following the induction dose.

A “dosage regimen” or “dosing regimen” includes a treatment regimenbased on a determined set of doses. In one embodiment, the inventiondescribes a dosage regimen for the treatment of Crohn's disease, whereinD2E7 is first administered as an induction dose and then administered intreatment doses which are lower than that of the induction dose.

The term “dosing”, as used herein, refers to the administration of asubstance (e.g., an anti-TNFα antibody) to achieve a therapeuticobjective (e.g., the treatment of a TNFα-associated disorder).

The terms “biweekly dosing regimen”, “biweekly dosing”, and “biweeklyadministration”, as used herein, refer to the time course ofadministering a substance (e.g., an anti-TNFα antibody) to a subject toachieve a therapeutic objective (e.g., the treatment of aTNFα-associated disorder). The biweekly dosing regimen is not intendedto include a weekly dosing regimen. Preferably, the substance isadministered every 9-19 days, more preferably, every 11-17 days, evenmore preferably, every 13-15 days, and most preferably, every 14 days.

The term “combination” as in the phrase “a first agent in combinationwith a second agent” includes co-administration of a first agent and asecond agent, which for example may be dissolved or intermixed in thesame pharmaceutically acceptable carrier, or administration of a firstagent, followed by the second agent, or administration of the secondagent, followed by the first agent. The present invention, therefore,includes methods of combination therapeutic treatment and combinationpharmaceutical compositions.

The term “concomitant” as in the phrase “concomitant therapeutictreatment” includes administering an agent in the presence of a secondagent. A concomitant therapeutic treatment method includes methods inwhich the first, second, third, or additional agents areco-administered. A concomitant therapeutic treatment method alsoincludes methods in which the first or additional agents areadministered in the presence of a second or additional agents, whereinthe second or additional agents, for example, may have been previouslyadministered. A concomitant therapeutic treatment method may be executedstep-wise by different actors. For example, one actor may administer toa subject a first agent and a second actor may to administer to thesubject a second agent, and the administering steps may be executed atthe same time, or nearly the same time, or at distant times, so long asthe first agent (and additional agents) are after administration in thepresence of the second agent (and additional agents). The actor and thesubject may be the same entity (e.g., human).

The term “combination therapy”, as used herein, refers to theadministration of two or more therapeutic substances, e.g., an anti-TNFαantibody and another drug, such as a DMARD or NSAID. The other drug(s)may be administered concomitant with, prior to, or following theadministration of an anti-TNFα antibody.

The term “TNFα-mediated condition” or “TNFα-related disorder” refers toa local and/or systemic physiological disorder where TNFα is a primarymediator leading to the manifestation of the disorder.

The term “kit” as used herein refers to a packaged product comprisingcomponents with which to administer the TNFα antibody of the inventionfor treatment of a TNFα-related disorder. The kit preferably comprises abox or container that holds the components of the kit. The box orcontainer is affixed with a label or a Food and Drug Administrationapproved protocol. The box or container holds components of theinvention which are preferably contained within plastic, polyethylene,polypropylene, ethylene, or propylene vessels. The vessels can becapped-tubes or bottles. The kit can also include instructions foradministering the TNFα antibody of the invention. In one embodiment thekit of the invention includes the formulation comprising the humanantibody D2E7, as described in PCT/IB03/04502 and U.S. application Ser.No. 10/222,140.

Various aspects of the invention are described in further detail herein.

II. TNFα Inhibitors of the Invention

This invention provides a multiple-variable dose method of treating aTNFα-related disorder in which the administration of a TNFα inhibitor isbeneficial. In one embodiment, these methods include administration ofisolated human antibodies, or antigen-binding portions thereof, thatbind to human TNFα with high affinity and a low off rate, and have ahigh neutralizing capacity. Preferably, the human antibodies of theinvention are recombinant, neutralizing human anti-hTNFα antibodies. Themost preferred recombinant, neutralizing antibody of the invention isreferred to herein as D2E7, also referred to as HUMIRA® and adalimumab(the amino acid sequence of the D2E7 VL region is shown in SEQ ID NO: 1;the amino acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2).The properties of D2E7 (HUMIRA®) have been described in Salfeld et al.,U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015, which are eachincorporated by reference herein. Other examples of TNFα inhibitorsinclude chimeric and humanized murine anti-hTNFα antibodies which haveundergone clinical testing for treatment of rheumatoid arthritis (seee.g., Elliott, M. J., et al. (1994) Lancet 344:1125-1127; Elliot, M. J.,et al. (1994) Lancet 344:1105-1110; Rankin, E. C., et al. (1995) Br. J.Rheumatol. 34:334-342).

In one embodiment, the multiple-variable dose method of the inventionincludes the administration of D2E7 antibodies and antibody portions,D2E7-related antibodies and antibody portions, and other humanantibodies and antibody portions with equivalent properties to D2E7,such as high affinity binding to hTNFα with low dissociation kineticsand high neutralizing capacity. In one embodiment, the inventionprovides multiple-variable dose treatment with an isolated humanantibody, or an antigen-binding portion thereof, that dissociates fromhuman TNFα with a K_(d) of 1×10⁻⁸ M or less and a K_(off) rate constantof 1×10⁻³ s⁻¹ or less, both determined by surface plasmon resonance, andneutralizes human TNFα cytotoxicity in a standard in vitro L929 assaywith an IC₅₀ of 1×10⁻⁷ M or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, dissociates from humanTNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less, or even more preferably, witha K_(off) of 1×10⁻⁴ s⁻¹ or less. More preferably, the isolated humanantibody, or antigen-binding portion thereof, neutralizes human TNFαcytotoxicity in a standard in vitro L929 assay with an IC₅₀ of 1×10⁻⁸ Mor less, even more preferably with an IC₅₀ of 1×10⁻⁹ M or less and stillmore preferably with an IC₅₀ of 1×10⁻¹⁰ M or less. In a preferredembodiment, the antibody is an isolated human recombinant antibody, oran antigen-binding portion thereof.

It is well known in the art that antibody heavy and light chain CDR3domains play an important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in another aspect, the inventionpertains to multiple-variable dose methods of treating a TNFα-relateddisorder in which the TNFα activity is detrimental by administeringhuman antibodies that have slow dissociation kinetics for associationwith hTNFα and that have light and heavy chain CDR3 domains thatstructurally are identical to or related to those of D2E7. Position 9 ofthe D2E7 VL CDR3 can be occupied by Ala or Thr without substantiallyaffecting the K_(off) Accordingly, a consensus motif for the D2E7 VLCDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ IDNO: 3). Additionally, position 12 of the D2E7 VH CDR3 can be occupied byTyr or Asn, without substantially affecting the K_(off) Accordingly, aconsensus motif for the D2E7 VH CDR3 comprises the amino acid sequence:V-S-Y-L-S-T-A-S-S-L-D (Y/N) (SEQ ID NO: 4). Moreover, as demonstrated inExample 2 of U.S. Pat. No. 6,090,382, the CDR3 domain of the D2E7 heavyand light chains is amenable to substitution with a single alanineresidue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantiallyaffecting the K_(off) Still further, the skilled artisan will appreciatethat, given the amenability of the D2E7 VL and VH CDR3 domains tosubstitutions by alanine, substitution of other amino acids within theCDR3 domains may be possible while still retaining the low off rateconstant of the antibody, in particular substitutions with conservativeamino acids. Preferably, no more than one to five conservative aminoacid substitutions are made within the D2E7 VL and/or VH CDR3 domains.More preferably, no more than one to three conservative amino acidsubstitutions are made within the D2E7 VL and/or VH CDR3 domains.Additionally, conservative amino acid substitutions should not be madeat amino acid positions critical for binding to hTNFα. Positions 2 and 5of the D2E7 VL CDR3 and positions 1 and 7 of the D2E7 VH CDR3 appear tobe critical for interaction with hTNFα and thus, conservative amino acidsubstitutions preferably are not made at these positions (although analanine substitution at position 5 of the D2E7 VL CDR3 is acceptable, asdescribed above) (see U.S. Pat. No. 6,090,382).

Accordingly, in another embodiment, the invention providesmultiple-variable dose methods of treating a TNFα-related disorder bythe administration of an isolated human antibody, or antigen-bindingportion thereof. The antibody or antigen-binding portion thereofpreferably contains the following characteristics:

a) dissociates from human TNFα with a K_(off) rate constant of 1×10⁻³s⁻¹ or less, as determined by surface plasmon resonance;

b) has a light chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alaninesubstitution at position 1, 4, 5, 7 or 8 or by one to five conservativeamino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9;

c) has a heavy chain CDR3 domain comprising the amino acid sequence ofSEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single alaninesubstitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to fiveconservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9,10, 11 and/or 12.

More preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 5×10⁻⁴ s⁻¹ or less. Evenmore preferably, the antibody, or antigen-binding portion thereof,dissociates from human TNFα with a K_(off) of 1×10⁻⁴ s⁻¹ or less.

In yet another embodiment, the invention provides multiple-variable dosemethods of treating a TNFα-related disorder by the administration of anisolated human antibody, or antigen-binding portion thereof. Theantibody or antigen-binding portion thereof preferably contains a lightchain variable region (LCVR) having a CDR3 domain comprising the aminoacid sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a singlealanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chainvariable region (HCVR) having a CDR3 domain comprising the amino acidsequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a singlealanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.Preferably, the LCVR further has a CDR2 domain comprising the amino acidsequence of SEQ ID NO: 5 (i.e., the D2E7 VL CDR2) and the HCVR furtherhas a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6(i.e., the D2E7 VH CDR2). Even more preferably, the LCVR further hasCDR1 domain comprising the amino acid sequence of SEQ ID NO: 7 (i.e.,the D2E7 VL CDR1) and the HCVR has a CDR1 domain comprising the aminoacid sequence of SEQ ID NO: 8 (i.e., the D2E7 VH CDR1). The frameworkregions for VL preferably are from the V_(K)I human germline family,more preferably from the A20 human germline Vk gene and most preferablyfrom the D2E7 VL framework sequences shown in FIGS. 1A and 1B of U.S.Pat. No. 6,090,382. The framework regions for VH preferably are from theV_(H)3 human germline family, more preferably from the DP-31 humangermline VH gene and most preferably from the D2E7 VH frameworksequences shown in FIGS. 2A and 2B of U.S. Pat. No. 6,090,382.

Accordingly, in another embodiment, the invention providesmultiple-variable dose methods of treating a TNFα-related disorder bythe administration of an isolated human antibody, or antigen-bindingportion thereof. The antibody or antigen-binding portion thereofpreferably contains a light chain variable region (LCVR) comprising theamino acid sequence of SEQ ID NO: 1 (i.e., the D2E7 VL) and a heavychain variable region (HCVR) comprising the amino acid sequence of SEQID NO: 2 (i.e., the D2E7 VH). In certain embodiments, the antibodycomprises a heavy chain constant region, such as an IgG1, IgG2, IgG3,IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chainconstant region is an IgG1 heavy chain constant region or an IgG4 heavychain constant region. Furthermore, the antibody can comprise a lightchain constant region, either a kappa light chain constant region or alambda light chain constant region. Preferably, the antibody comprises akappa light chain constant region. Alternatively, the antibody portioncan be, for example, a Fab fragment or a single chain Fv fragment.

In still other embodiments, the invention provides multiple-variabledose methods of treating a TNFα-related disorder in which theadministration of an anti-TNFα antibody is beneficial administration ofan isolated human antibody, or an antigen-binding portions thereof. Theantibody or antigen-binding portion thereof preferably containsD2E7-related VL and VH CDR3 domains, for example, antibodies, orantigen-binding portions thereof, with a light chain variable region(LCVR) having a CDR3 domain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ IDNO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 orwith a heavy chain variable region (HCVR) having a CDR3 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQID NO: 35.

In another embodiment, the TNFα inhibitor of the invention is etanercept(described in WO 91/03553 and WO 09/406476), infliximab (described inU.S. Pat. No. 5,656,272), CDP571 (a humanized monoclonal anti-TNF-alphaIgG4 antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibodyfragment), D2E7 (a human anti-TNF mAb), soluble TNF receptor Type I, ora pegylated soluble TNF receptor Type I (PEGs TNF-R1).

The TNFα antibody of the invention can be modified. In some embodiments,the TNFα antibody or antigen binding fragments thereof, is chemicallymodified to provide a desired effect. For example, pegylation ofantibodies and antibody fragments of the invention may be carried out byany of the pegylation reactions known in the art, as described, forexample, in the following references: Focus on Growth Factors 3:4-10(1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated byreference herein in its entirety). Preferably, the pegylation is carriedout via an acylation reaction or an alkylation reaction with a reactivepolyethylene glycol molecule (or an analogous reactive water-solublepolymer). A preferred water-soluble polymer for pegylation of theantibodies and antibody fragments of the invention is polyethyleneglycol (PEG). As used herein, “polyethylene glycol” is meant toencompass any of the forms of PEG that have been used to derivatizeother proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethyleneglycol.

Methods for preparing pegylated antibodies and antibody fragments of theinvention will generally comprise the steps of (a) reacting the antibodyor antibody fragment with polyethylene glycol, such as a reactive esteror aldehyde derivative of PEG, under conditions whereby the antibody orantibody fragment becomes attached to one or more PEG groups, and (b)obtaining the reaction products. It will be apparent to one of ordinaryskill in the art to select the optimal reaction conditions or theacylation reactions based on known parameters and the desired result.

Pegylated antibodies and antibody fragments may generally be used totreat TNFα-related disorders of the invention by administration of theTNFα antibodies and antibody fragments described herein. Generally thepegylated antibodies and antibody fragments have increased half-life, ascompared to the nonpegylated antibodies and antibody fragments. Thepegylated antibodies and antibody fragments may be employed alone,together, or in combination with other pharmaceutical compositions.

In yet another embodiment of the invention, TNFα antibodies or fragmentsthereof can be altered wherein the constant region of the antibody ismodified to reduce at least one constant region-mediated biologicaleffector function relative to an unmodified antibody. To modify anantibody of the invention such that it exhibits reduced binding to theFc receptor, the immunoglobulin constant region segment of the antibodycan be mutated at particular regions necessary for Fc receptor (FcR)interactions (see e.g., Canfield, S. M. and S. L. Morrison (1991) J.Exp. Med. 173:1483-1491; and Lund, J. et al. (1991) J. of Immunol.147:2657-2662). Reduction in FcR binding ability of the antibody mayalso reduce other effector functions which rely on FcR interactions,such as opsonization and phagocytosis and antigen-dependent cellularcytotoxicity.

An antibody or antibody portion of the invention can be derivatized orlinked to another functional molecule (e.g., another peptide orprotein). Accordingly, the antibodies and antibody portions of theinvention are intended to include derivatized and otherwise modifiedforms of the human anti-hTNFα antibodies described herein, includingimmunoadhesion molecules. For example, an antibody or antibody portionof the invention can be functionally linked (by chemical coupling,genetic fusion, noncovalent association or otherwise) to one or moreother molecular entities, such as another antibody (e.g., a bispecificantibody or a diabody), a detectable agent, a cytotoxic agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, e.g., to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

An antibody, or antibody portion, of the invention can be prepared byrecombinant expression of immunoglobulin light and heavy chain genes ina host cell. To express an antibody recombinantly, a host cell istransfected with one or more recombinant expression vectors carrying DNAfragments encoding the immunoglobulin light and heavy chains of theantibody such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium the antibodies can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss et al.

To express D2E7 or a D2E7-related antibody, DNA fragments encoding thelight and heavy chain variable regions are first obtained. These DNAscan be obtained by amplification and modification of germline light andheavy chain variable sequences using the polymerase chain reaction(PCR). Germline DNA sequences for human heavy and light chain variableregion genes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson, I.M., et al. (1992) “The Repertoire of Human Germline V_(H) SequencesReveals about Fifty Groups of V_(H) Segments with DifferentHypervariable Loops” J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al.(1994) “A Directory of Human Germ-line V₇₈ Segments Reveals a StrongBias in their Usage” Eur. J. Immunol. 24:827-836; the contents of eachof which are expressly incorporated herein by reference). To obtain aDNA fragment encoding the heavy chain variable region of D2E7, or aD2E7-related antibody, a member of the V_(H)3 family of human germlineVH genes is amplified by standard PCR. Most preferably, the DP-31 VHgermline sequence is amplified. To obtain a DNA fragment encoding thelight chain variable region of D2E7, or a D2E7-related antibody, amember of the V_(K)I family of human germline VL genes is amplified bystandard PCR. Most preferably, the A20 VL germline sequence isamplified. PCR primers suitable for use in amplifying the DP-31 germlineVH and A20 germline VL sequences can be designed based on the nucleotidesequences disclosed in the references cited supra, using standardmethods.

Once the germline VH and VL fragments are obtained, these sequences canbe mutated to encode the D2E7 or D2E7-related amino acid sequencesdisclosed herein. The amino acid sequences encoded by the germline VHand VL DNA sequences are first compared to the D2E7 or D2E7-related VHand VL amino acid sequences to identify amino acid residues in the D2E7or D2E7-related sequence that differ from germline. Then, theappropriate nucleotides of the germline DNA sequences are mutated suchthat the mutated germline sequence encodes the D2E7 or D2E7-relatedamino acid sequence, using the genetic code to determine whichnucleotide changes should be made. Mutagenesis of the germline sequencesis carried out by standard methods, such as PCR-mediated mutagenesis (inwhich the mutated nucleotides are incorporated into the PCR primers suchthat the PCR product contains the mutations) or site-directedmutagenesis.

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained (by amplification and mutagenesis of germline VH and VL genes,as described above), these DNA fragments can be further manipulated bystandard recombinant DNA techniques, for example to convert the variableregion genes to full-length antibody chain genes, to Fab fragment genesor to a scFv gene. In these manipulations, a VL- or VH-encoding DNAfragment is operatively linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operatively linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CHL CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene,the VH-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but most preferably is a kappaconstant region.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VLsequences can be expressed as a contiguous single-chain protein, withthe VL and VH regions joined by the flexible linker (see e.g., Bird etal. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains, obtained asdescribed above, are inserted into expression vectors such that thegenes are operatively linked to transcriptional and translationalcontrol sequences. In this context, the term “operatively linked” isintended to mean that an antibody gene is ligated into a vector suchthat transcriptional and translational control sequences within thevector serve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vector or, more typically, both genesare inserted into the same expression vector. The antibody genes areinserted into the expression vector by standard methods (e.g., ligationof complementary restriction sites on the antibody gene fragment andvector, or blunt end ligation if no restriction sites are present).Prior to insertion of the D2E7 or D2E7-related light or heavy chainsequences, the expression vector may already carry antibody constantregion sequences. For example, one approach to converting the D2E7 orD2E7-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the antibodychain from a host cell. The antibody chain gene can be cloned into thevector such that the signal peptide is linked in-frame to the aminoterminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding antibody genes are introducedinto mammalian host cells, the antibodies are produced by culturing thehost cells for a period of time sufficient to allow for expression ofthe antibody in the host cells or, more preferably, secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods.

Host cells can also be used to produce portions of intact antibodies,such as Fab fragments or scFv molecules. It is understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of this invention. Recombinant DNA technology mayalso be used to remove some or all of the DNA encoding either or both ofthe light and heavy chains that is not necessary for binding to hTNFα.The molecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention. In addition,bifunctional antibodies may be produced in which one heavy and one lightchain are an antibody of the invention and the other heavy and lightchain are specific for an antigen other than hTNFα by crosslinking anantibody of the invention to a second antibody by standard chemicalcrosslinking methods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr-CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells areculture to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.

Recombinant human antibodies of the invention in addition to D2E7 or anantigen binding portion thereof, or D2E7-related antibodies disclosedherein can be isolated by screening of a recombinant combinatorialantibody library, preferably a scFv phage display library, preparedusing human VL and VH cDNAs prepared from mRNA derived from humanlymphocytes. Methodologies for preparing and screening such librariesare known in the art. In addition to commercially available kits forgenerating phage display libraries (e.g., the Pharmacia RecombinantPhage Antibody System, catalog no. 27-9400-01; and the StratageneSurfZAP™ phage display kit, catalog no. 240612), examples of methods andreagents particularly amenable for use in generating and screeningantibody display libraries can be found in, for example, Ladner et al.U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619;Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCTPublication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

In a preferred embodiment, to isolate human antibodies with highaffinity and a low off rate constant for hTNFα, a murine anti-hTNFαantibody having high affinity and a low off rate constant for hTNFα(e.g., MAK 195, the hybridoma for which has deposit number ECACC 87050801) is first used to select human heavy and light chain sequenceshaving similar binding activity toward hTNFα, using the epitopeimprinting methods described in Hoogenboom et al., PCT Publication No.WO 93/06213. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described in McCafferty et al.,PCT Publication No. WO 92/01047, McCafferty et al., Nature (1990)348:552-554; and Griffiths et al., (1993) EMBO J 12:725-734. The scFvantibody libraries preferably are screened using recombinant human TNFαas the antigen.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for hTNFα binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve theaffinity and/or lower the off rate constant for hTNFα binding, the VLand VH segments of the preferred VL/VH pair(s) can be randomly mutated,preferably within the CDR3 region of VH and/or VL, in a processanalogous to the in vivo somatic mutation process responsible foraffinity maturation of antibodies during a natural immune response. Thisin vitro affinity maturation can be accomplished by amplifying VH and VLregions using PCR primers complimentary to the VH CDR3 or VL CDR3,respectively, which primers have been “spiked” with a random mixture ofthe four nucleotide bases at certain positions such that the resultantPCR products encode VH and VL segments into which random mutations havebeen introduced into the VH and/or VL CDR3 regions. These randomlymutated VH and VL segments can be rescreened for binding to hTNFα andsequences that exhibit high affinity and a low off rate for hTNFαbinding can be selected.

Following screening and isolation of an anti-hTNFα antibody of theinvention from a recombinant immunoglobulin display library, nucleicacid encoding the selected antibody can be recovered from the displaypackage (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention (e.g., linked to nucleic acid encoding additionalimmunoglobulin domains, such as additional constant regions). To expressa recombinant human antibody isolated by screening of a combinatoriallibrary, the DNA encoding the antibody is cloned into a recombinantexpression vector and introduced into a mammalian host cells, asdescribed in further detail in above.

Methods of isolating human antibodies with high affinity and a low offrate constant for hTNFα are also described in U.S. Pat. Nos. 6,090,382,6,258,562, and 6,509,015, each of which is incorporated by referenceherein.

III. Uses of the TNFα Inhibitors of the Invention

The invention provides a multiple-variable dose method for inhibitingTNFα activity in a subject suffering from a disorder in which TNFαactivity is detrimental. TNFα has been implicated in the pathophysiologyof a wide variety of disorders (see e.g., Moeller, A., et al. (1990)Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.; EuropeanPatent Publication No. 260 610 B1 by Moeller, A.). TNFα has beenimplicated in the pathophysiology of a wide variety of a TNFα-relateddisorders including sepsis, infections, autoimmune diseases, transplantrejection and graft-versus-host disease (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A., et al.Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey, K. J. andCerami, A. (1994) Annu. Rev. Med. 45:491-503). The invention providesmultiple-variable dose methods for inhibiting TNFα activity in a subjectsuffering from a TNFα-related disorder, which method comprisesadministering to a subject an initial induction dose and subsequentlyadministering a treatment dose of an antibody, antibody portion, orother TNFα inhibitor, such that TNFα activity is inhibited. Preferably,the TNFα is human TNFα and the subject is a human subject. In oneembodiment, the TNFα inhibitor is D2E7, also referred to as HUMIRA®(adalimumab).

As used herein, the term “a disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders inwhich the presence of TNFα in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which TNFαactivity is detrimental is a disorder in which inhibition of TNFαactivity is expected to alleviate the symptoms and/or progression of thedisorder. Such disorders may be evidenced, for example, by an increasein the concentration of TNFα in a biological fluid of a subjectsuffering from the disorder (e.g., an increase in the concentration ofTNFα in serum, plasma, synovial fluid, etc. of the subject), which canbe detected, for example, using an anti-TNFα antibody as describedabove. There are numerous examples of disorders in which TNFα activityis detrimental. The use of TNFα inhibitors, including antibodies andantibody portions, of the invention in the treatment of specificdisorders using a multiple-variable dose therapy is discussed furtherbelow:

A. Sepsis

Tumor necrosis factor has an established role in the pathophysiology ofsepsis, with biological effects that include hypotension, myocardialsuppression, vascular leakage syndrome, organ necrosis, stimulation ofthe release of toxic secondary mediators and activation of the clottingcascade (see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S.Pat. No. 5,231,024 to Moeller et al.; European Patent Publication No.260 610 B1 by Moeller, A.; Tracey, K. J. and Cerami, A. (1994) Annu.Rev. Med. 45:491-503; Russell, D and Thompson, R. C. (1993) Curr. Opin.Biotech. 4:714-721). The multiple-variable dose methods of the inventioncan be used to treat sepsis in any of its clinical settings, includingseptic shock, endotoxic shock, gram negative sepsis and toxic shocksyndrome.

Furthermore, to treat sepsis, an anti-hTNFα antibody, or antibodyportion, of the invention can be coadministered with one or moreadditional therapeutic agents that may further alleviate sepsis, such asan interleukin-1 inhibitor (such as those described in PCT PublicationNos. WO 92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g.,PCT Publication No. WO 93/11793) or an antagonist of platelet activatingfactor (see e.g., European Patent Application Publication No. EP 374510). Other combination therapies including multiple-variable dosetherapies for the treatment of sepsis are discussed further insubsection IV. In a preferred embodiment, an anti-TNFα antibody orantibody portion is administered to a human subject within a subgroup ofsepsis patients having a serum or plasma concentration of IL-6 above 500pg/ml, and more preferably 1000 pg/ml, at the time of treatment (see PCTPublication No. WO 95/20978 by Daum, L., et al.).

B. Autoimmune Diseases

Tumor necrosis factor has been implicated in playing a role in thepathophysiology of a variety of autoimmune diseases. For example, TNFαhas been implicated in activating tissue inflammation and causing jointdestruction in rheumatoid arthritis (see e.g., Moeller, A., et al.(1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller et al.;European Patent Publication No. 260 610 B1 by Moeller, A.; Tracey andCerami, supra; Arend, W. P. and Dayer, J-M. (1995) Arth. Rheum.38:151-160; Fava, R. A., et al. (1993) Clin. Exp. Immunol. 94:261-266).TNFα also has been implicated in promoting the death of islet cells andin mediating insulin resistance in diabetes (see e.g., Tracey andCerami, supra; PCT Publication No. WO 94/08609). TNFα also has beenimplicated in mediating cytotoxicity to oligodendrocytes and inductionof inflammatory plaques in multiple sclerosis (see e.g., Tracey andCerami, supra). TNFα also has been implicated in mediating cytotoxicityto oligodendrocytes and induction of inflammatory plaques in multiplesclerosis (see e.g., Tracey and Cerami, supra). Chimeric and humanizedmurine anti-hTNFα antibodies have undergone clinical testing fortreatment of rheumatoid arthritis (see e.g., Elliott, M. J., et al.(1994) Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.34:334-342).

TNFα inhibitors, including human antibodies, and antibody portions suchas D2E7, may be used in a multiple-variable dose method to treatautoimmune diseases, in particular those associated with inflammation.Examples of such autoimmune conditions include rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy,multiple sclerosis, autoimmune diabetes, autoimmune uveitis andnephrotic syndrome. Other examples of autoimmune conditions includemultisystem autoimmune diseases and autoimmune hearing loss.

Typically, the antibody, or antibody portion, is administeredsystemically, although for certain disorders, local administration ofthe antibody or antibody portion at a site of inflammation may bebeneficial (e.g., local administration in the joints in rheumatoidarthritis or topical application to diabetic ulcers, alone or incombination with a cyclohexane-ylidene derivative as described in PCTPublication No. WO 93/19751). TNFα inhibitors, including humanantibodies, and antibody portions such as D2E7, also can be administeredwith one or more additional therapeutic agents useful in themultiple-variable dose treatment of autoimmune diseases, as discussedfurther in subsection IV.

In one embodiment of the invention, a human TNFα antibody is used inmultiple-variable dose therapy to treat autoimmune disorders such aslupus. Lupus is has been shown to be associated with TNF activity(Shvidel et al. (2002) Hematol J. 3:32; Studnicka-Benke et al. (1996) BrJ Rheumatol. 35:1067). The term “lupus” as used herein refers to achronic, inflammatory autoimmune disorder called lupus erythematosusthat may affect many organ systems including the skin, joints andinternal organs. Lupus is a general term which includes a number ofspecific types of lupus, including systemic lupus, lupus nephritis, andlupus cerebritis. In systemic lupus (SLE), the body's natural defensesare turned against the body and rogue immune cells attack the body'stissues. Antibodies may be produced that can react against the body'sblood cells, organs, and tissues. This reaction leads to immune cellsattacking the affected systems, producing a chronic disease. Lupusnephritis, also referred to as lupus glomerular disease, is kidneydisorder that is usually a complication of SLE, and is characterized bydamage to the glomerulus and progressive loss of kidney function. Lupuscerebritis refers to another complication of SLE, which is inflammationof the brain and/or central nervous system.

Another autoimmune disease which can be treated using themultiple-variable dose treatment of the invention is Crohn's disease,which is described in more detail below in the Intestinal DisordersSection.

C. Infectious Diseases

Tumor necrosis factor has been implicated in mediating biologicaleffects observed in a variety of infectious diseases. For example, TNFαhas been implicated in mediating brain inflammation and capillarythrombosis and infarction in malaria. TNFα also has been implicated inmediating brain inflammation, inducing breakdown of the blood-brainbarrier, triggering septic shock syndrome and activating venousinfarction in meningitis. TNFα also has been implicated in inducingcachexia, stimulating viral proliferation and mediating central nervoussystem injury in acquired immune deficiency syndrome (AIDS).Accordingly, antibodies, and antibody portions, directed against TNF,can be used in multiple-variable dose treatment of infectious diseases,including bacterial meningitis (see e.g., European Patent ApplicationPublication No. EP 585 705), cerebral malaria, AIDS and AIDS-relatedcomplex (ARC) (see e.g., European Patent Application Publication No. EP230 574), as well as cytomegalovirus infection secondary totransplantation (see e.g., Fietze et al. (1994) Transplantation 58:675).The antibodies, and antibody portions, of the invention, also can beused to alleviate symptoms associated with infectious diseases,including fever and myalgias due to infection (such as influenza) andcachexia secondary to infection (e.g., secondary to AIDS or ARC).

D. Transplantation

Tumor necrosis factor has been implicated as a key mediator of allograftrejection and graft versus host disease (GVHD) and in mediating anadverse reaction that has been observed when the rat antibody OKT3,directed against the T cell receptor CD3 complex, is used to inhibitrejection of renal transplants (see e.g., Eason et al. (1995)Transplantation 59:300; Suthanthiran and Strom (1994) New Engl. J. Med.331:365). Accordingly, the antibodies, and antibody portions, of theinvention, can be used to inhibit transplant rejection usingmultiple-variable dose treatment, including rejections of allografts andxenografts and to inhibit GVHD. Although the antibody or antibodyportion may be used alone, more preferably it is used in combinationwith one or more other agents that inhibit the immune response againstthe allograft or inhibit GVHD. For example, in one embodiment, anantibody or antibody portion of the invention is used in combinationwith OKT3 to inhibit OKT3-induced reactions. In another embodiment, anantibody or antibody portion of the invention is used in combinationwith one or more antibodies directed at other targets involved inregulating immune responses, such as the cell surface molecules CD25(interleukin-2 receptor-α), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45,CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment,an antibody or antibody portion of the invention is used in combinationwith one or more general immunosuppressive agents, such as cyclosporin Aor FK506.

E. Malignancy

Tumor necrosis factor has been implicated in inducing cachexia,stimulating tumor growth, enhancing metastatic potential and mediatingcytotoxicity in malignancies. Accordingly, antibodies, and antibodyportions, which directed against TNF, can be used in themultiple-variable dose treatment of malignancies, wherein treatmentinhibits tumor growth or metastasis and/or alleviates cachexia secondaryto malignancy. The antibody, or antibody portion, may be administeredsystemically or locally to the tumor site.

F. Pulmonary Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofadult respiratory distress syndrome (ARDS), including stimulatingleukocyte-endothelial activation, directing cytotoxicity to pneumocytesand inducing vascular leakage syndrome. The multiple-variable dosemethods of the invention can be used to treat various pulmonarydisorders, including adult respiratory distress syndrome, usingmultiple-variable dose treatment (see e.g., PCT Publication No. WO91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonarysarcoidosis, pulmonary fibrosis and silicosis. The antibody, or antibodyportion, may be administered systemically or locally to the lungsurface, for example as an aerosol. An antibody, or antibody portion,also can be administered with one or more additional therapeutic agentsuseful in the multiple-variable dose treatment of pulmonary disorders,as discussed further in subsection IV.

Other examples of pulmonary disorders in which TNFα has been implicatedin the pathophysiology include idiopathic interstitial lung disease andchronic obstructive airway disorders (see e.g., Piquet et al. (1989) JExp Med. 170:655; Whyte et al. (2000) Am J Respir Crit Care Med.162:755; Anticevich et al. (1995) Eur J Pharmacol. 284:221). Theinvention further provides methods for treating TNFα activity in asubject suffering from such a pulmonary disorder, which method comprisesadministering to the subject an antibody, antibody portion, or otherTNFα inhibitor using a multiple variable dose regimen such that TNFαactivity in the subject suffering from idiopathic interstitial lungdisease or a chronic obstructive airway disorder is inhibited. Examplesof idiopathic interstitial lung diseases and chronic obstructive airwaydisorders in which TNFα activity is detrimental are discussed furtherbelow.

1. Idiopathic Interstitial Lung Disease

In one embodiment, the TNFα antibody of the invention is used inmultiple-variable dose treatment regimen to treat subjects who have anidiopathic interstitial lung disease. The term “idiopathic pulmonaryfibrosis” or “IPF” refers to a group of disorders characterized byinflammation and eventually scarring of the deep lung tissues, leadingto shortness of breath. The scarring of the alveoli (air sacs) and theirsupporting structures (the interstitium) in IPF eventually leads to aloss of the functional alveolar units and a reduction of the transfer ofoxygen from air to blood. IPF is also referred to as diffuse parenchymallung disease; alveolitis; cryptogenic fibrosing alveolitis (CFA);idiopathic pulmonary pneumonitis (IPP); and usual interstitialpneumonitis (UIP). IPF is often used synonymously with UIP (“IPF/UIP”)because UIP is the most common cellular pattern seen in the pathologicdiagnosis of IPF.

Idiopathic interstitial lung diseases affect the lungs in three ways:first, the lung tissue is damaged in some known or unknown way; second,the walls of the air sacs in the lung become inflamed; and finally,scarring (or fibrosis) begins in the interstitium (or tissue between theair sacs), and the lung becomes stiff. Examples of idiopathicinterstitial lung diseases include idiopathic pulmonary fibrosis (IPF).Tumor necrosis factor has been implicated in the pathophysiology ofidiopathic pulmonary fibrosis (IPF) (see Piquet et al. (1989) J Exp Med.170:655; Whyte et al. (2000) Am J Respir Crit Care Med 162:755 Corbettet al. (2002) Am J Respir Crit Care Med. 165:690). For example, it hasbeen found that IPF patients have increased levels of TNF expression inmacrophages and in type II epithelial cells (Piquet et al. (1993) Am JPathol 143:651; Nash et al. (1993) Histopathology 22:343; Zhang et al.(1993) J Immunol 150:4188). Certain genetic polymorphisms are alsoassociated with increased TNF expression, and are implicated in playinga role in IPF and silicosis (Whyte et al., supra; Corbett et al.,supra).

Patients with IPF often exhibit certain symptoms, including a dry cough,chest pain, and/or shortness of breath. Commonly used drugs for thetreatment of IPF are prednisone and cytoxan, although only a fraction ofpatients improve with continued use of these drugs (American ThoracicSociety (2000) Am. J. Respir. Crit. Care Med. 161:646). Oxygenadministration and transplantation of the lung are other choices fortreatment. In one embodiment, antibodies used in the multiple-variabledose methods of the invention may be used in combination with anothertherapeutic agent, for example oxygen, for the treatment of idiopathicpulmonary fibrosis.

Examples of animal models used to study idiopathic interstitial lungdisease and chronic obstructive airway disorders include ovalbumin (OVA)induced allergic asthma mice and cigarette smoke induced chronicobstructive pulmonary disease mice (see Hessel et al. (1995) Eur JPharmacol. 293:401; Keast et al. (1981) J. Pathol. 135:249).

2. Chronic obstructive airway disorder

In one embodiment, a TNFα antibody is used in multiple-variable dosetreatment regimen to treat a subject who has a chronic obstructiveairflow disorder. In these diseases, airflow obstruction may be chronicand persistent or episodic and recurrent. Airflow obstruction is usuallydetermined by forced expiratory spirometry, which is the recording ofexhaled volume against time during a maximal expiration. In a subjectwho does not have an obstructed airflow, a full forced expirationusually takes between 3 and 4 seconds. In a patient with chronicobstructive airflow disorder, wherein airflow is obstructed, it usuallytakes up to 15 to 20 seconds and may be limited by breath-holding time.The normal forced expiratory volume in the first second of expiration(FEV₁) is easily measured and accurately predicted on the basis of age,sex, and height. The ratio of FEV₁ to forced vital capacity (FEV₁/FVC)normally exceeds 0.75. Recording airflow against volume during forcedexpiration and a subsequent forced inspiration—the flow-volume loop—isalso useful, mainly for distinguishing upper from lower airwaynarrowing. Examples of chronic obstructive airway disorders aredescribed below.

a. Asthma

Tumor necrosis factor has been implicated in the pathophysiology ofasthma, (Anticevich et al. (1995) Eur J Pharmacol. 284:221; Thomas etal. 1995. Am J Respir Crit Care Med. 152:76; Thomas and Heywood (2002)Thorax. 57:774). For example, acute asthma attacks have been found to beassociated with pulmonary neutrophilia and elevated BAL TNF levels(Ordonez et al. (2000) Am J Respir Crit Care Med 161:1185). It has beenfound that the severity of asthma symptoms correlates with endotoxinlevels in house dust. In rats, anti-TNF antibodies reducedendotoxin-induced airway changes (Kips et al. (1992) Am Rev Respir Dis145:332).

The term “asthma” as used herein, refers to a disorder in whichinflammation of the airways causes airflow into and out of the lungs tobe restricted. Asthma is also referred to as bronchial asthma, exerciseinduced asthma—bronchial, and reactive airways disease (RAD). In someinstances, asthma is associated with allergies and/or is familial.Asthma includes a condition which is characterized by widespreadfluctuations in the diameter or caliber of bronchial airways over shortperiods of time, resulting in changes in lung function. The resultingincreased resistance to air flow produces symptoms in the affectedsubject, including breathlessness (dyspnea), chest constriction or“tightness,” and wheezing.

Patients with asthma are characterized according to NIH guidelines, aredescribed as mild intermittent, mild persistent, moderate persistent,and severe persistent (see NAEPP Expert Panel Report Guidelines for theDiagnosis and Management of Asthma-Update on Selected Topics 2002. JACI2002; 110: S141-S209; Guidelines for the Diagnosis and Management ofAsthma. NIH Publication 97-4051, July 1997). Patients diagnosed withmoderate persistent asthma are often treated with inhaledcorticosteroids. Patients diagnosed with severe persistent asthma areoften treated with high dose inhaled corticosteroids and p.o.corticosteroids.

b. Chronic Obstructive Pulmonary Disease (CORD)

Tumor necrosis factor has been implicated in the pathophysiology ofchronic obstructive pulmonary disease, (Keatings (2000) Chest. 118:971;Sakao et al. (2001) Am J Respir Crit Care Med. 163:420; Sakao et al.(2002) Chest. 122:416). The term “chronic obstructive pulmonary disease”or “COPD” as used interchangeably herein, refers to a group of lungdiseases characterized by limited airflow with variable degrees of airsack enlargement and lung tissue destruction. The term COPD includeschronic bronchitis (mucous hypersecretion with goblet cell submucosalgland hyperplasia), chronic obstructive bronchitis, or emphysema(destruction of airway parenchyma), or combinations of these conditions.Emphysema and chronic bronchitis are the most common forms of chronicobstructive pulmonary disease. COPD is defined by irreversible airflowobstruction.

In COPD, chronic inflammation leads to fixed narrowing of small airwaysand lung parenchyma and alveolar wall destruction (emphysema). This ischaracterized by increased numbers of alveolar macrophages, neutrophils,and cytotoxic T lymphocytes, and the release of multiple inflammatorymediators (lipids, chemokines, cytokines, growth factors). Thisinflammation leads to fibrosis with a narrowing of the small airways andlung parenchymal destruction. There is also a high level of oxidativestress, which may amplify this inflammation.

G. Intestinal Disorders

Tumor necrosis factor has been implicated in the pathophysiology ofinflammatory bowel disorders including Crohn's disease (see e.g., Tracyet al. (1986) Science 234:470; Sun et al. (1988) J. Clin. Invest.81:1328; MacDonald et al. (1990) Clin. Exp. Immunol. 81:301). Chimericmurine anti-hTNFα antibodies have undergone clinical testing fortreatment of Crohn's disease (van Dullemen et al. (1995)Gastroenterology 109:129). The invention includes a multiple-variabledose regimen comprising administering a TNFα inhibitor to treatintestinal disorders, such as idiopathic inflammatory bowel disease,using human antibodies, or antigen-binding fragments thereof. Idiopathicinflammatory bowel disease includes two syndromes, Crohn's disease andulcerative colitis. In one embodiment, the multiple-variable doseregimen of the invention is also used to treat disorders oftenassociated with IBD and Crohn's disease. The term “inflammatory boweldisorder (IBD)-related disorder” or “Crohn's disease-related disorder,”as used interchangeably herein, is used to describe conditions andcomplications commonly associated with IBD and Crohn's disease.

The invention includes a multiple-variable dose regimen comprisingadministering a TNFα inhibitor to treat Crohn's disease. The treatmentof Crohn's disease is based on location, extent, and severity ofdisease. Pharmacologic interventions include anti-inflammatory agents(aminosalicylates and corticosteroids) and immunomodulatory agents(azathioprine and 6-mercaptopurine[6-MP], cyclosporine, methotrexate[MTX], antibiotic agents, and biologic agents). C-reactive protein (CRP)and erythrocyte sedimentation rate (ESR) levels reflect non-specificacute phase reactions. Endoscopy is a primary means of diagnosingCrohn's disease. Radiologic features of Crohn's disease are shown bybarium examination includes mucosal edema, aphthous and linearulcerations, asymmetrical narrowing and strictures, and separation ofadjacent loops of bowel caused by mesenteric thickening. Abnormalitiesare focal and asymmetric. The primary histologic lesion is an aphthousulcer. Subjects with Crohn's disease can be evaluated using the Crohn'sDisease Activity Index (CDAI), which is a standard measure of theseverity of the disease with higher scores indicating more severedisease activity.

Examples of Crohn's disease-related disorders which can be treated usingthe methods of the invention include fistulas in the bladder, vagina,and skin; bowel obstructions; abscesses; nutritional deficiencies;complications from corticosteroid use; inflammation of the joints;erythem nodosum; pyoderma gangrenosum; and lesions of the eye. Otherdisorders commonly associated with Crohn's disease includeCrohn's-related arthralgias, fistulizing Crohn's, indeterminant colitis,and pouchitis.

H. Cardiac Disorders

The multiple-variable dose methods of the invention also can be used totreat in of various cardiac or coronary disorders, including ischemia ofthe heart (see e.g., European Patent Application Publication No. EP 453898) and heart insufficiency (weakness of the heart muscle)(see e.g.,PCT Publication No. WO 94/20139). TNFα has also been implicated in thepathophysiology of restenosis (see e.g., Clausell et al. (1994), supra;Medall et al. (1997) Heart 78:273).

As used herein, the term “a cardiac disorder in which TNFα activity isdetrimental” is intended to include coronary and cardiovascular diseasesin which the presence of TNFα in a subject suffering from the disorderhas been shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder, including cardiovascular disorders, e.g.,restenosis. The term “cardiovascular disorder” or “coronary disorder” asused interchangeably herein, refers to any disease, disorder, or stateinvolving the cardiovascular system, e.g., the heart, the blood vessels,and/or the blood. A coronary disorder is generally characterized by anarrowing of the blood vessels that supply blood and oxygen to the heart(coronary arteries). Coronary disease may result from the build up offatty material and plaque. As the coronary arteries narrow, the flow ofblood to the heart can slow or stop. Coronary disorders of the inventioncan apply to any abnormality of an artery, whether structural,histological, biochemical or any other abnormality. An example ofcoronary heart disease is restenosis. In one embodiment, a coronarydisorder refers to any disease, disorder, or state involving thecardiovascular system excluding ischemia of the heart and heartinsufficiency.

Coronary disorders in which TNFα activity is detrimental often resultfrom a blockage in an artery. Such a blockage can be caused by a clot,which usually forms in a coronary artery that has been previouslynarrowed from changes usually related to atherosclerosis. For example,if the atherosclerotic plaque inside the arterial wall cracks, it cantrigger the formation of a thrombus, or clot. Such disorders may beevidenced, for example, by an increase in the concentration of TNFα in abiological fluid of a subject suffering from the disorder (e.g., anincrease in the concentration of TNFα in serum, plasma, synovial fluid,etc. of the subject), which can be detected, for example, using ananti-TNFα antibody as described above. A coronary disorder can be alsocaused by an imbalance in arterial pressure, a malfunction of the heart,or an occlusion of a blood vessel, e.g., by a thrombus. Coronarydisorders includes both coronary artery disease and peripheral vasculardisease.

There are numerous examples of cardiac disorders in which TNFα activityis detrimental, including restenosis. The use of the antibodies,antibody portions, and other TNFα inhibitors in multiple-variable doseregimens for treatment of specific coronary disorders is discussedfurther below. In certain embodiments, a antibody, antibody portion, orother TNFα inhibitor is administered to the subject in combination withanother therapeutic agent, as described below.

The invention provides a multiple-variable dose method for inhibitingTNFα activity in a subject with a cardiac disorder. The inventionprovides multiple-variable dose methods for inhibiting or decreasingTNFα activity in a subject with a coronary disorder, comprisingadministering to the subject an antibody, or antibody portion, or otherTNFα inhibitor of the invention such that TNFα activity in the subjectis inhibited or decreased. Preferably, the TNFα is human TNFα and thesubject is a human subject. Alternatively, the subject can be a mammalexpressing a TNFα with which an antibody of the invention cross-reacts.Still further the subject can be a mammal into which has been introducedhTNFα (e.g., by administration of hTNFα or by expression of an hTNFαtransgene). An antibody of the invention can be administered to a humansubject for therapeutic purposes.

Moreover, an antibody of the invention can be administered to anon-human mammal expressing a TNFα with which the antibody cross-reacts(e.g., a primate, pig or mouse) for veterinary purposes or as an animalmodel of human disease. Regarding the latter, such animal models may beuseful for evaluating the multiple-variable dose therapeutic efficacy(e.g., testing of dosages and time courses of administration). Commonlyused animal models for studying coronary disorders, includingrestenosis, include the rat or mouse carotid artery ligation model andthe carotid artery injury model (Ferns et al. (1991) Science 253:1129;Clowes et al. (1983) Lab. Invest. 49:208; Lindner et al. (1993) CircRes. 73:792). In the carotid artery ligation model, arterial blood flowis disrupted by ligation of the vessel near the distal bifurnation. Asdescribed in Clowes et al., the carotid artery injury model is performedsuch that the common carotid artery is denuded of endothelium by theintraluminal passage of a balloon catheter introduced through theexternal carotid artery. At 2 weeks, the carotid artery is markedlynarrowed due to smooth muscle cell constriction, but between 2 and 12weeks the intimal doubles in thickness leading to a decrease in luminalsize. Any of these models can be used to determine the potentialtherapeutic action of the TNFα antibodies of the invention in theprevention and treatment of restenosis in humans.

The invention includes multiple-variable dose regimen for treatment ofcardiovascular disorders in which TNFα activity is detrimental, whereininhibition of TNFα activity is expected to alleviate the symptoms and/orprogression of the coronary disease or to prevent the coronary disease.Subjects suffering from or at risk of developing coronary disorders canbe identified through clinical symptoms. Clinical symptoms in coronarydisease often include chest pain, shortness of breath, weakness,fainting spells, alterations in consciousness, extremity pain,paroxysmal nocturnal dyspnea, transient ischemic attacks and other suchphenomena experienced by the patient. Clinical signs of coronary diseasecan also include EKG abnormalities, altered peripheral pulses, arterialbruits, abnormal heart sounds, rates and wheezes, jugular venousdistention, neurological alterations and other such findings discernedby the clinician. Coronary disorders may also be evidenced, for example,by an increase in the concentration of TNFα in a biological fluid of asubject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject).

Examples of a cardiovascular disorder include, but are not limited to,coronary artery disease, angina pectoris, myocardial infarction,cardiovascular tissue damage caused by cardiac arrest, cardiovasculartissue damage caused by cardiac bypass, cardiogenic shock, andhypertension, atherosclerosis, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias, and cardiomyopathies. The use ofthe antibodies, antibody portions, and other TNFα inhibitors inmultiple-variable dose regimens for treatment of specific cardiovasculardiseases are discussed further below. In certain embodiments, theantibody, antibody portion, or other TNFα inhibitor is administered tothe subject in combination with another therapeutic agent, as describedbelow in section IV.

1. Restenosis

The term “restenosis” as used herein refers to the recurrence ofstenosis, which is the narrowing or constriction of an artery.Restenosis often occurs as a preocclusive lesion that develops followinga reconstructive procedure in a diseased blood vessel. The term is notonly applied to the recurrence of a pre-existing stenosis, but also topreviously normal vessels that become partially occluded followingvascular bypass. In another embodiment, the invention provides a methodof treating restenosis comprising administering the antibody, or antigenbinding portion thereof, of the invention to a subject who has or is atrisk of developing restenosis.

TNFα has been implicated in the pathophysiology of restenosis (see Zhouet al. (2002) Atherosclerosis. 161:153; Javed et al. (2002) Exp and MolPathol 73:104). For example, in the murine wire carotid model, TNF−/−mice demonstrated a seven-fold reduction in initial hyperplasia comparedto wild type mice (Zimmerman et al. (2002) Am J Phsiol Regul Integr CompPhysiol 283:R505). Restenosis can occur as the result of any type ofvascular reconstruction, whether in the coronary vasculature or in theperiphery (Colburn and Moore (1998) Myointimal Hyperplasia pp. 690-709in Vascular Surgery: A Comprehensive Review Philadelphia: Saunders). Forexample, studies have reported symptomatic restenosis rates of 30-50%following coronary angioplasties (see Berk and Harris (1995) Adv.Intern. Med. 40:455). After carotid endarterectomies, as a furtherexample, 20% of patients studied had a luminal narrowing greater than50% (Clagett et al. (1986) J. Vasc. Surg. 3:10). Restenosis is evidencedin different degrees of symptomatology which accompany preocclusivelesions in different anatomical locations, due to a combination offactors including the nature of the vessels involved, the extent ofresidual disease, and local hemodynamics.

“Stenosis,” as used herein refers to a narrowing of an artery as seen inocclusive disorder or in restenosis. Stenosis can be accompanied bythose symptoms reflecting a decrease in blood flow past the narrowedarterial segment, in which case the disorder giving rise to the stenosisis termed a disease (i.e., occlusive disease or restenosis disease).Stenosis can exist asymptomatically in a vessel, to be detected only bya diagnostic intervention such as an angiography or a vascular labstudy.

The multiple-variable dose method of the invention can be used to treata subject suffering from or at risk of developing restenosis. A subjectat risk of developing restenosis includes a subject who has undergonePTCA. The subject may have also had a stent inserted to preventrestenosis. The TNFα antibody can be used alone or in combination with astent to prevent the re-occurrence of stenosis in a subject sufferingfrom cardiovascular disease.

2. Congestive Heart Failure

TNFα has been implicated in the pathophysiology of congestive heartfailure (see Zhou et al. (2002) Atherosclerosis 161:153). Serum levelsof TNFα are elevated in patients with congestive heart failure in amanner which is directly proportional to the severity of the disease(Levine et al. (1990) N Engl J Med 323:236; Torre-Amione et al. (1996) JAm Coll Cardiol 27:1201). In addition, inhibitors of TNFα have also beenshown to improve congestive heart failure symptoms (Chung et al. (2003)Circulation 107:3133).

As used herein, the term “congestive heart failure” includes a conditioncharacterized by a diminished capacity of the heart to supply the oxygendemands of the body. Symptoms and signs of congestive heart failureinclude diminished blood flow to the various tissues of the body,accumulation of excess blood in the various organs, e.g., when the heartis unable to pump out the blood returned to it by the great veins,exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheraledema resulting from left ventricular dysfunction. Congestive heartfailure may be acute or chronic. The manifestation of congestive heartfailure usually occurs secondary to a variety of cardiac or systemicdisorders that share a temporal or permanent loss of cardiac function.Examples of such disorders include hypertension, coronary arterydisease, valvular disease, and cardiomyopathies, e.g., hypertrophic,dilative, or restrictive cardiomyopathies.

A “subject who has or is suffering from congestive heart failure” is asubject who has a disorder involving a clinical syndrome of diverseetiologies linked by the common denominator of impaired heart pumping inwhich the heart cannot pump blood commensurate with the requirements ofthe metabolizing tissues, or can do so only from an elevated fillingpressure. A “subject at risk of developing congestive heart failure” isa subject who has a propensity of developing congestive heart failurebecause of certain factors affecting the cardiovascular system of thesubject. It is desirable to reduce the risk of or prevent thedevelopment of congestive heart failure in these subjects. The phrase“with congestive heart failure” includes patients who are at risk ofsuffering from this condition relative to the general population, eventhough they may not have suffered from it yet, by virtue of exhibitingrisk factors. For example, a patient with untreated hypertension may nothave suffered from congestive heart failure, but is at risk because ofhis or her hypertensive condition. In one embodiment of the invention,the antibody D2E7 is used to treat a subject at risk of developingcongestive heart failure using multiple-variable dose treatment.

3. Acute Coronary Syndromes

TNFα has been implicated in the pathophysiology of acute coronarysyndromes (see Libby (1995) Circulation 91:2844). Acute coronarysyndromes include those disorders wherein the subject experiences paindue to a blood flow restriction resulting in not enough oxygen reachingthe heart. Studies have found that TNFα plays a role in acute coronarysyndromes. For example, in a novel rat heterotropic cardiactransplantation-coronary ligation model capable of inducing myocardialinfarction in the absence of downstream hemodynamic effects,administration of chimeric soluble TNF receptor (sTNFR) abolishedtransient LV remodeling and dysfunction (Nakamura, et al. (2003) J.Cardiol. 41:41). It was also found that direct injection of an sTNFRexpression plasmid to the myocardium, resulted in a reduction in theinfarction size in acute myocardial infarction (AMI) experimental rats(Sugano et al. (2002) FASEB J 16:1421).

In one embodiment, a TNFα antibody is used in a multiple-variable dosemethod for the treatment or prevention of an acute coronary syndrome ina subject, wherein the acute coronary syndrome is a myocardialinfarction or angina.

As used herein, the term “myocardial infarction” or “MI” refers to aheart attack. A myocardial infarction involves the necorsis or permanentdamage of a region of the heart due to an inadequate supply of oxygen tothat area. This necrosis is typically caused by an obstruction in acoronary artery from either atherosclerosis or an embolis. MIs which aretreated by the TNFα antibody of the invention include both Q-wave andnon-Q-wave myocardial infarction. Most heart attacks are caused by aclot that blocks one of the coronary arteries (the blood vessels thatbring blood and oxygen to the heart muscle). For example, a clot in thecoronary artery interrupts the flow of blood and oxygen to the heartmuscle, leading to the death of heart cells in that area. The damagedheart muscle permanently loses its ability to contract, and theremaining heart muscle needs to compensate for it. An MI can also becaused by overwhelming stress in the individual.

The term “angina” refers to spasmodic, choking, or suffocative pain, andespecially as denoting angina pectoris which is a paroxysmal thoracicpain due, most often, to anoxia of the myocardium. Angina includes bothvariant angina and exertional angina. A subject having angina hasischemic heart disease which is manifested by sudden, severe, pressingsubstemal pain that often radiates to the left shoulder and along theleft arm. TNFα has been implicated in angina, as TNFα levels areupregulated in patients with both MI and stable angina (Balbay et al.(2001) Angiology 52109).

4. Artherosclerosis

“Atherosclerosis” as used herein refers to a condition in which fattymaterial is deposited along the walls of arteries. This fatty materialthickens, hardens, and may eventually block the arteries.Atherosclerosis is also referred to arteriosclerosis, hardening of thearteries, and arterial plaque buildup. Polyclonal antibodies directedagainst TNFα have been shown to be effective at neutralizing TNFαactivity resulting in inflammation and restenosis in the rabbitatherosclerotic model (Zhou et al., supra). Accordingly, a TNFα antibodycan be used to treat or prevent subjects afflicted with or at risk ofhaving atherosclerosis using the multiple-variable dose method of theinvention.

5. Cardiomyopathy

The term “cardiomyopathy” as used herein is used to define diseases ofthe myocardium wherein the heart muscle or myocardium is weakened,usually resulting in inadequate heart pumping. Cardiomyopathy can becaused by viral infections, heart attacks, alcoholism, long-term, severehypertension (high blood pressure), or by autoimmune causes.

In approximately 75-80% of heart failure patients coronary arterydisease is the underlying cause of the cardiomyopathy and is designated“ischemic cardiomyopathy.” Ischemic cardiomyopathy is caused by heartattacks, which leave scars in the heart muscle or myocardium. Theaffected myocardium is then unable to contribute to the heart pumpingfunction. The larger the scars or the more numerous the heart attacks,the higher the chance there is of developing ischemic cardiomyopathy.

Cardiomyopathies that are not attributed to underlying coronary arterydisease, and are designated “non-ischemic cardiomyopathies.”Non-ischemic cardiomyopathies include, but are not limited to idiopathiccardiomyopathy, hypertrophic cardiomyopathy, alcoholic cardiomyopathy,dilated cardiomyopathy, peripartum cardiomyopathy, and restrictivecardiomyopathy.

I. Spondyloarthropathies

TNFα has been implicated in the pathophysiology of a wide variety ofdisorders, including inflammatory diseases such as spondyloarthopathies(see e.g., Moeller et al. (1990) Cytokine 2:162; U.S. Pat. No.5,231,024; European Patent Publication No. 260 610). The inventionprovides multiple-variable dose methods for inhibiting TNFα activity ina subject suffering from a spondyloarthropathy, which method comprisesadministering to the subject an antibody, antibody portion, or otherTNFα inhibitor initially in an induction dose, followed by a treatmentdose, such that TNFα activity in the subject suffering from aspondyloarthropathy is inhibited.

As used herein, the term “spondyloarthropathy” or“spondyloarthropathies” is used to refer to any one of several diseasesaffecting the joints of the spine, wherein such diseases share commonclinical, radiological, and histological features. A number ofspondyloarthropathies share genetic characteristics, i.e. they areassociated with the HLA-B27 allele. In one embodiment, the termspondyloarthropathy is used to refer to any one of several diseasesaffecting the joints of the spine, excluding ankylosing spondylitis,wherein such diseases share common clinical, radiological, andhistological features. Examples of spondyloarthropathies includeankylosing spondylitis, psoriatic arthritis/spondylitis, enteropathicarthritis, reactive arthritis or Reiter's syndrome, and undifferentiatedspondyloarthropathies. Examples of animal models used to studyspondyloarthropathies include ank/ank transgenic mice, HLA-B27transgenic rats (see Taurog et al. (1998) The Spondylarthritides.Oxford:Oxford University Press).

The multiple-variable dose methods of the invention can also be used totreat subjects who are at risk of developing a spondyloarthropathy usingmultiple-variable dose methods. Examples of subjects who are at risk ofhaving spondyloarthropathies include humans suffering from arthritis.Spondyloarthropathies can be associated with other forms of arthritis,including rheumatoid arthritis. In one embodiment of the invention,antibodies are used in multiple-variable dose methods to treat a subjectwho suffers from a spondyloarthropathy associated with rheumatoidarthritis. Examples of spondyloarthropathies which can be treated with aTNFα antibody using the multiple-variable dose method of the inventionare described below:

1. Ankylosing Spondylitis (AS)

Tumor necrosis factor has been implicated in the pathophysiology ofankylosing spondylitis (see Verjans et al. (1991) Arthritis Rheum.34:486; Verjans et al. (1994) Clin Exp Immunol. 97:45; Kaijtzel et al.(1999) Hum Immunol. 60:140). Ankylosing spondylitis (AS) is aninflammatory disorder involving inflammation of one or more vertebrae.AS is a chronic inflammatory disease that affects the axial skeletonand/or peripheral joints, including joints between the vertebrae of thespine and sacroiliac joints and the joints between the spine and thepelvis. AS can eventually cause the affected vertebrae to fuse or growtogether. Spondyarthropathies, including AS, can be associated withpsoriatic arthritis (PsA) and/or inflammatory bowel disease (IBD),including ulcerative colitis and Crohn's disease.

Early manifestations of AS can be determined by radiographic tests,including CT scans and MRI scans. Early manifestations of AS ofteninclude scroiliitis and changes in the sacroliac joints as evidenced bythe blurring of the cortical margins of the subchrondral bone, followedby erosions and sclerosis. Fatigue has also been noted as a commonsymptom of AS (Duffy et al. (2002) ACR 66th Annual Scientific MeetingAbstract). Accordingly, multiple-variable dose methods comprisingadministering an antibody, or antigen-binding fragment thereof, of theinvention can be used to treat AS.

In one embodiment, the multiple-variable dose method of the invention isused to treat a spondyloarthropathy associated with IBD, including AS.AS is often treated with nonsteroidal anti-inflammatory medications(NSAIDs), such as aspirin or indomethacin. Accordingly, a TNFα antibodyused in the multiple-variable dose method of the invention may also beadministered in combination with agents commonly used to reduceinflammation and pain commonly associated with ankylosing spondylitis.

2. Psoriatic Arthritis

Tumor necrosis factor has been implicated in the pathophysiology ofpsoriatic arthritis (PsA) (Partsch et al. (1998) Ann Rheum Dis. 57:691;Ritchlin et al. (1998) J Rheumatol. 25:1544). As referred to herein,psoriatic arthritis or psoriasis associated with the skin, refers tochronic inflammatory arthritis which is associated with psoriasis, whichis a common chronic skin condition that causes red patches on the body.About 1 in 20 individuals with psoriasis will develop arthritis alongwith the skin condition, and in about 75% of cases, psoriasis precedesthe arthritis. PsA exhibits itself in a variety of ways, ranging frommild to severe arthritis, wherein the arthritis usually affects thefingers and the spine. When the spine is affected, the symptoms aresimilar to those of ankylosing spondylitis, as described above. The TNFαantibody, or antigen-binding fragment thereof, of the invention can beused in multiple-variable dose treatment of PsA.

PsA is sometimes associated with arthritis mutilans. Arthritis mutilansrefers to a disorder which is characterized by excessive bone erosionresulting in a gross, erosive deformity which mutilates the joint. Inone embodiment, the multiple-variable dose method of the invention canbe used to treat arthritis mutilans.

3. Reactive Arthritis/Reiter's Syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofreactive arthritis, which is also referred to as Reiter's syndrome(Braun et al. (1999) Arthritis Rheum. 42(10):2039). Reactive arthritis(ReA) refers to arthritis which complicates an infection elsewhere inthe body, often following enteric or urogenital infections. ReA is oftencharacterized by certain clinical symptoms, including inflammation ofthe joints (arthritis), urethritis, conjunctivitis, and lesions of theskin and mucous membranes. In addition, ReA can occurs followinginfection with a sexually transmitted disease or dysenteric infection,including chlamydia, campylobacter, salmonella, or yersinia.Accordingly, the multiple-variable dose method of the invention can beused to treat ReA using the multiple-variable dose method of theinvention.

4. Undifferentiated Spondyloarthropathies

In one embodiment, multiple-variable dose methods of the invention ofthe invention are used to treat subjects suffering from undifferentiatedspondyloarthropathies (see Zeidler et al. (1992) Rheum Dis Clin NorthAm. 18:187). Other terms used to describe undifferentiatedspondyloarthropathies include seronegative oligoarthritis andundifferentiated oligoarthritis. Undifferentiated spondyloarthropathies,as used herein, refers to a disorder wherein the subject demonstratesonly some of the symptoms associated with a spondyloarthropathy. Thiscondition is usually observed in young adults who do not have IBD,psoriasis, or the classic symptoms of AS or Reiter's syndrome. In someinstances, undifferentiated spondyloarthropathies may be an earlyindication of AS. In one embodiment, the multiple-variable dose methodof the invention comprises administering different doses of a TNFαantibody, or antigen-binding fragment thereof, to treat undifferentiatedspondyloarthropathies.

J. Metabolic Disorders

TNFα has been implicated in the pathophysiology of a wide variety ofdisorders, including metabolic disorders, such as diabetes and obesity(Spiegelman and Hotamisligil (1993) Cell 73:625; Chu et al. (2000) Int JObes Relat Metab Disord. 24:1085; Ishii et al. (2000) Metabolism.49:1616). The term “metabolic disorder,” as used herein, refers todiseases or disorders which affect how the body processes substancesneeded to carry out physiological functions. Examples of metabolicdisorders include, but are not limited to, diabetes and obesity. In oneembodiment of the invention, the term “metabolic disorder” is used torefer to disorders which affect how the body processes substances neededto carry out physiological functions, excluding autoimmune diabetes.

The invention provides multiple-variable dose methods for inhibitingTNFα activity in a subject suffering from such a metabolic disorder,which method comprises administering to the subject an induction dosefollowed by a treatment dose of an antibody, antibody portion, or otherTNFα inhibitor such that TNFα activity in the subject suffering from ametabolic disorder is inhibited. TNFα antibodies can also be used totreat subjects who are at risk of developing a metabolic disorder usingthe multiple-variable dose regimen of the invention.

Metabolic disorders are often associated with arthritis, includingrheumatoid arthritis. In one embodiment, a TNFα inhibitor, such as anantibody, is used in a multiple-variable dose regimen in a subject whosuffers from a metabolic disorder associated with rheumatoid arthritis.In another embodiment, the multiple-variable dose treatment of theinvention comprises administering a TNFα antibody to treat disordersassociated with diabetes or obesity.

Examples of animal models for evaluating the efficacy of amultiple-variable dose regimen using a TNFα antibody for the treatmentof a metabolic disorder include NOD transgenic mice, Akita mice, NSYtransgenic mice and ob/ob mice (see Baeder et al. (1992) Clin ExpImmunol. 89:174; Haseyama et al. (2002) Tohoku J Exp Med. 198:233;Makino et al. (1980): Exp. Anim. 29:1; Kolb (1987) Diabetes/MetabolismReviews 3:751; Hamada et al. (2001) Metabolism. 50:1282; Coleman, (1978)Diabetologia, 14:141; Bailey et al. (1982) Int. J. Obesity 6:11).Examples of animal models used to study vasculitis includes the mouseHSV model (Behcet's disease), the mouse L. casei model (Kawasaki'sdisease), and the mouse ANCA model (Kawasaki's disease). Other models ofvasculitis include the McHS-lpr/lpr strain (Nose et al. (1996) Am. IPath. 149:1763) and the SCG/Kj strain of mice (Kinjoh et al. (1993)Proc. Natl. Acad. Sci., USA 90:3413). These mice strains spontaneouslydevelop crescentic glomerulonephritis and necrotizing vasculitis of thesmall arteries and arterioles of the spleen, stomach, heart, uterus andovaries. These animals develop hypergammaglobulinemia and ANCAautoantibodies that react with myeloperoxidase (MPO). Additionally,immunization of rats with human MPO results in ANCA-associatednecrotizing crescentic glomerulonephritis (Brouwer et al. (1993) J. Exp.Med. 177:905).

Metabolic disorders affect how the body processes substances needed tocarry out physiological functions. A number of metabolic disorders ofthe invention share certain characteristics, i.e. they are associatedthe insulin resistance, lack of ability to regulate blood sugar, weightgain, and increase in body mass index. Examples of metabolic disordersinclude diabetes and obesity. Examples of diabetes include type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations, diabetic macrovasculopathy, and obesity.Examples of metabolic disorders which can be treated usingmultiple-variable dose methods comprising administration of a TNFαantibody are described in more detail below:

1. Diabetes

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetes. (see e.g., Navarro et al. (2003) Am J Kidney Dis. 42:53;Daimon et al. (2003) Diabetes Care. 26:2015; Zhang et al. (1999) JTongji Med Univ. 19:203; Barbieri et al. (2003) Am J Hypertens. 16:537)For example, TNFα is implicated in the pathophysiology for insulinresistance. It has been found that serum TNF levels in patients withgastrointestinal cancer correlates with insulin resistance (see e.g.,McCall et al. (1992) Br. J. Surg. 79:1361).

The term “diabetes” or “diabetic disorder” or “diabetes mellitus,” asused interchangeably herein, refers to a disease which is marked byelevated levels of sugar (glucose) in the blood. Diabetes can be causedby too little insulin (a chemical produced by the pancreas to regulateblood sugar), resistance to insulin, or both. Diabetes includes the twomost common types of the disorder, namely type I diabetes and type IIdiabetes, which both result from the body's inability to regulateinsulin. Insulin is a hormone released by the pancreas in response toincreased levels of blood sugar (glucose) in the blood.

The term “type 1 diabetes,” as used herein, refers to a chronic diseasethat occurs when the pancreas produces too little insulin to regulateblood sugar levels appropriately. Type 1 diabetes is also referred to asinsulin-dependent diabetes mellitus, IDMM, juvenile onset diabetes, anddiabetes—type I. Type 1 diabetes represents is the result of aprogressive autoimmune destruction of the pancreatic β-cells withsubsequent insulin deficiency.

The term “type 2 diabetes,” refers to a chronic disease that occurs whenthe pancreas does not make enough insulin to keep blood glucose levelsnormal, often because the body does not respond well to the insulin.Type 2 diabetes is also referred to as noninsulin-dependent diabetesmellitus, NDDM, and diabetes—type II

Diabetes is can be diagnosed by the administration of a glucosetolerance test. Clinically, diabetes is often divided into several basiccategories. Primary examples of these categories include, autoimmunediabetes mellitus, non-insulin-dependent diabetes mellitus (type 1NDDM), insulin-dependant diabetes mellitus (type 2 IDDM), non-autoimmunediabetes mellitus, non-insulin-dependant diabetes mellitus (type 2NIDDM), and maturity-onset diabetes of the young (MODY). A furthercategory, often referred to as secondary, refers to diabetes broughtabout by some identifiable condition which causes or allows a diabeticsyndrome to develop. Examples of secondary categories include, diabetescaused by pancreatic disease, hormonal abnormalities, drug- orchemical-induced diabetes, diabetes caused by insulin receptorabnormalities, diabetes associated with genetic syndromes, and diabetesof other causes. (see e.g., Harrison's (1996) 14^(th) ed., New York,McGraw-Hill).

Diabetes is often treated with diet, insulin dosages, and variousmedications described herein. Accordingly, a TNFα antibody may also beadministered in combination with agents commonly used to treat metabolicdisorders and pain commonly associated with diabetes in themultiple-variable dose method of the invention.

In addition, the phrase “disorders associated with diabetes,” as usedherein, refers to conditions and other diseases which are commonlyassociated with or related to diabetes. Example of disorders associatedwith diabetes include, for example, hyperglycemia, hyperinsulinaemia,hyperlipidaemia, insulin resistance, impaired glucose metabolism,obesity, diabetic retinopathy, macular degeneration, cataracts, diabeticnephropathy, glomerulosclerosis, diabetic neuropathy, erectiledysfunction, premenstrual syndrome, vascular restenosis, ulcerativecolitis, coronary heart disease, hypertension, angina pectoris,myocardial infarction, stroke, skin and connective tissue disorders,foot ulcerations, metabolic acidosis, arthritis, and osteoporosis. Inone embodiment the multiple-variable dose methods of the invention canbe used to treat disorders associated with diabetes.

Diabetes manifests itself in the foregoing categories and can causeseveral complications that are discussed in the following sections.Accordingly, the antibody, or antigen-binding fragment thereof, of theinvention can be used to treat diabetes. In one embodiment, a TNFαantibody, or antigen-binding fragment thereof, is used to treat diabetesassociated with the above identified categories using themultiple-variable dose method of the invention. In another embodiment,the invention includes multiple-variable dose regimens comprisingadministering a TNFα antibody to treat disorders associated withdiabetes. Diabetes manifests itself in many complications and conditionsassociated with diabetes, including the following catagories:

a. Diabetic Neuropathy and Peripheral Neuropathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic neuropathy and peripheral neuropathy. (See Benjafield et al.(2001) Diabetes Care. 24:753; Qiang et al. (1998) Diabetologia. 41:1321;Pfeiffer et al. (1997) Horm Metab Res. 29:111).

The term “neuropathy,” also referred to as nerve damage-diabetic, asused herein, refers to a common complication of diabetes in which nervesare damaged as a result of hyperglycemia (high blood sugar levels). Avariety of diabetic neuropathies are recognized, such as distalsensorimotror polyneuropathy, focal motor neuropathy, and autonomicneuropathy.

The term “peripheral neuropathy,” also known as peripheral neuritis anddiabetic neuropathy, as used herein, refers to the failure of the nervesto carry information to and from the brain and spinal cord. Peripheralneuropathy produces symptoms such as pain, loss of sensation, and theinability to control muscles. In some cases, the failure of nerves tocontrol blood vessels, intestinal function, and other organs results inabnormal blood pressure, digestion, and loss of other basic involuntaryprocesses. Peripheral neuropathy may involve damage to a single nerve ornerve group (mononeuropathy) or may affect multiple nerves(polyneuropathy).

Neuropathies that affect small myelinated and unmyelinated fibers of thesympathetic and parasympathetic nerves are known as “peripheralneuropathies.” Furthermore, the related disorder of peripheralneuropathy, also known as peripheral neuritis and diabetic neuropathy,refers to the failure of the nerves to carry information to and from thebrain and spinal cord. This produces symptoms such as pain, loss ofsensation, and the inability to control muscles. In some cases, failureof nerves controlling blood vessels, intestinal function, and otherorgans results in abnormal blood pressure, digestion, and loss of otherbasic involuntary processes. Peripheral neuropathy may involve damage toa single nerve or nerve group (mononeuropathy) or may affect multiplenerves (polyneuropathy).

The term “diabetic neuropathy” refers to a common complication ofdiabetes in which nerves are damaged as a result of hyperglycemia (highblood sugar levels). Diabetic neuropathy is also referred to asneuropathy and nerve damage-diabetic. A variety of diabetic neuropathiesare recognized, such as distal sensorimotror polyneuropathy, focal motorneuropathy, and autonomic neuropathy.

b. Diabetic Retinopathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic retinopthy (Scholz et al. (2003) Trends Microbiol. 11:171). Theterm “diabetic retinopathy” as used herein, refers to progressive damageto the eye's retina caused by long-term diabetes. Diabetic retinopathy,includes proliferative retinopathy. Proliferative neuropathy in turnincludes includes neovascularization, pertinal hemmorrhave and retinaldetachement.

In advanced retinopathy, small vessels proliferate on the surface of theretina. These blood vessels are fragile, tend to bleed and can causeperetinal hemorrhages. The hemorrhage can obscure vision, and as thehemorrhage is resorbed fibrous tissue forms predisposing to retinaldetachments and loss of vision. In addition, diabetic retinopathyincludes proliferative retinopathy which includes neovascularization,pertinal hemmorrhave and retinal detachement. Diabetic retinopathy alsoincludes “background retinopathy” which involves changes occurring withthe layers of the retina.

c. Diabetic Ulcerations and Retinopathy Ulcerations

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic ulcerations, (see Lee et al. (2003) Hum Immunol. 64:614;Navarro et al. (2003) Am J Kidney Dis. 42:53; Daimon et al (2003)Diabetes Care. 26:2015; Zhang et al. (1999) J Tongji Med Univ. 19:203;Barbieri et al. (2003) Am J Hypertens. 16:537; Venn et al. (1993)Arthritis Rheum. 36:819; Westacott et al. (1994) J Rheumatol. 21:1710).

The term “diabetic ulcerations,” as used herein, refers to an ulcerwhich results as a complication of diabetes. An ulcer is a crater-likelesion on the skin or mucous membrane caused by an inflammatory,infectious, malignant condition, or metabolic disorder. Typicallydiabetic ulcers can be found on limbs and extremeties, more typicallythe feet. These ulcers, caused by diabetic conditions, such asneuropathy and a vacualr insuffciency, can lead to ischemia and poorwound healing. More extensive ulcerations may progress to ostemyelitis.Once ostemyelitis develops, it may be dificult to eradicate withantibotics alone and amputation maybe necessary.

The term “retinopathy ulcerations,” as used herein refers to an ulcerwhich causes or results in damages to the eye and the eye's retina.Retinopathy ulcerations may include conditions such has retinoathichemmorages.

d. Diabetic Macrovasculopathy

Tumor necrosis factor has been implicated in the pathophysiology ofdiabetic macrovasculopathy (Devaraj et al. (2000) Circulation. 102:191;Hattori et al. (2000) Cardiovasc Res. 46:188; Clausen et al. (1999)Cardiovasc Pathol. 8:145). The term “diabetic macrovasculopathy,” alsoreferred to as “macrovascular disease,” as used herein, refers to adisease of the blood vessels that results from diabetes. Diabeticmacrovasculopathy complication occurs when, for example, fat and bloodclots build up in the large blood vessels and stick to the vessel walls.Diabetic macrovasculopathies include diseases such as coronary disease,cerebrovascular disease, and peripheral vascular disease, hyperglycaemiaand cardiovascular disease, and strokes.

2. Obesity

Tumor necrosis factor has been implicated in the pathophysiology ofobesity (see e.g., Pihlajamaki J et al. (2003) Obes Res. 11:912;Barbieri et al. (2003) Am J Hypertens. 16:537; Tsuda et al. (2003) JNutr. 133:2125). The term “obesity” as used herein, refers to acondition in which the subject has an excess of body fat relative tolean body mass. In one embodiment, obesity refers to a condition inwhich an individual weighs at least about 20% or more over the maximumdesirable for their height. When an adult is more than 100 poundsoverweight, he or she is considered to be “morbidly obese.” In anotherembodiment, obesity is defined as a BMI (body mass index) over 30 kg/m2.Obesity increases a person's risk of illness and death due to diabetes,stroke, coronary artery disease, hypertension, high cholesterol, andkidney and gallbladder disorders. Obesity may also increase the risk forsome types of cancer, and may be a risk factor for the development ofosteoarthritis and sleep apnea. Obesity can be treated using themultiple-variable dose methods of the invention.

K. Anemia

TNFα has been implicated in the pathophysiology of a wide variety ofanemias (see e.g., Jongen-Lavrencic et al. (1997) J. Rheumatol. 24:1504;Demeter et al. (2002) Ann Hematol. 81:566; DiCato (2003) The Oncologist8 (suppl 1):19). The invention provides multiple-variable dose methodsfor inhibiting TNFα activity in a subject suffering from anemia, whichmethod comprises administering to the subject an induction dose followedby a treatment dose of a TNFα inhibitor, wherein the TNFα inhibitor isan antibody, antibody portion, such that TNFα activity in the subjectsuffering from anemia is inhibited. In one embodiment, the anemia isassociated with rheumatoid arthritis.

The term “anemia” as used herein, refers to an abnormally low number ofcirculating red cells or a decreased concentration of hemoglobin in theblood. Examples of anemia related to rheumatoid arthritis include, forexample, anemia of chronic disease, iron deficiency anemia, andautoimmune hemolytic anemia. In one embodiment, the invention provides amethod of treating anemias related to, for example, anemias related torheumatoid arthritis, anemias of infection and chronic inflammatorydiseases, iron deficiency anemia, autoimmune hemolytic anemia,myelophthisic anemia, aplastic anemia, hypoplastic anemia, pure red cellaplasia and anemia associated with renal failure or endocrine disorders,megaloblastic anemias, defects in heme or globin synthesis, anemiacaused by a structural defect in red blood cells, e.g., sickle-cellanemia, and anemias of unknown origins such as sideroblastic anemia,anemia associated with chronic infections such as malaria,trypanosomiasis, HIV, hepatitis virus or other viruses, andmyelophthisic anemias caused by marrow deficiencies.

Examples of animal models used to study anemia include rats inoculatedwith peptidolglycan-polysaccharide polymers (see Coccia et al., (2001)Exp Hematology. 29:1201-1209). Examples of animal models used to studypain are well known in the art, and include the rat sciatic nerveligation model, and the rat segmental spinal nerve ligation model (seeBennett and Zie, (1988) Pain. 33:87-107; Kim and Chung, (1992) Pain50:355-363).

L. Pain

TNFα has been implicated in the pathophysiology of a wide variety ofpain syndromes (see e.g., Sorkin et al. (1997) Neuroscience. 81:255;Huygen et al. (2002) Mediators Inflamm. 11:47; Parada et al. (2003) EurJ Neurosci. 17:1847). The term “pain” as used herein, refers to alltypes of pain. The term shall refer to acute and chronic pains, such asneuropathic pain and post-operative pain, chronic lower back pain,cluster headaches, herpes neuralgia, phantom limb pain, central pain,dental pain, opioid-resistant pain, visceral pain, surgical pain, boneinjury pain, pain during labor and delivery, pain resulting from burns,including sunburn, post partum pain, migraine, angina pain, andgenitourinary tract-related pain including cystitis. The term alsoincludes nociceptive pain or nociception.

The invention provides multiple-variable dose methods for inhibitingTNFα activity in a subject suffering from such a pain disorder, whichmethod comprises administering to the subject an induction dose followedby a treatment dose of an antibody, antibody portion, or other TNFαinhibitor such that TNFα activity in the subject suffering from pain isinhibited. Pain has been defined in a variety of ways, includingnociceptive pain and neuropathic pain. The most commonly experiencedform of pain may be defined as the effect of a stimulus on nerveendings, which results in the transmission of impulses to the cerebrum.Pain is also commonly associated with inflammatory disorders, including,for example, rheumatoid arthritis. In one embodiment, the antibody ofthe invention is used to treat a subject who suffers from painassociated with rheumatoid arthritis. Examples of pain disorders inwhich TNFα activity is detrimental are discussed further below.

1. Neuropathic Pain

Tumor necrosis factor has been implicated in the pathophysiology ofneuropathic pain (see Sommer (1999) Schmerz. 13:315; Empl et al., (2001)Neurology. 56:1371; Schafers et al. (2003) J Neurosci. 23:3028). As usedherein the term “neuropathic pain” refers to pain that results frominjury to a nerve, spinal cord, or brain, and often involves neuralsupersensitivity. Examples of neuropathic pain include chronic lowerback pain, pain associated with arthritis, cancer-associated pain,herpes neuralgia, phantom limb pain, central pain, opioid resistantneuropathic pain, bone injury pain, and pain during labor and delivery.Other examples of neuropathic pain include post-operative pain, clusterheadaches, dental pain, surgical pain, pain resulting from severe, forexample third degree, burns, post partum pain, angina pain,genitourinary tract related pain, and including cystitis.

Neuropathic pain is distinguished from nociceptive pain. Pain involvinga nociceptive mechanism usually is limited in duration to the period oftissue repair and generally is alleviated by available analgesic agentsor opioids (Myers (1995) Regional Anesthesia 20:173). Neuropathic paintypically is long-lasting or chronic and often develops days or monthsfollowing an initial acute tissue injury. Neuropathic pain can involvepersistent, spontaneous pain as well as allodynia, which is a painfulresponse to a stimulus that normally is not painful. Neuropathic painalso can be characterized by hyperalgesia, in which there is anaccentuated response to a painful stimulus that usually is trivial, suchas a pin prick. Unlike nociceptive pain, neuropathic pain generally isresistant to opioid therapy (Myers, supra, 1995). Accordingly, themultiple-variable dose methods of the invention can be used to treatneuropathic pain.

2. Nociceptive Pain

As used herein the term “nociceptive pain” refers to pain that istransmitted across intact neuronal pathways, i.e., pain caused by injuryto the body. Nociceptive pain includes somatic sensation and normalfunction of pain, and informs the subject of impending tissue damage.The nociceptive pathway exists for protection of the subject, e.g., thepain experienced in response to a burn). Nociceptive pain includes bonepain, visceral pain, and pain associated with soft tissue.

Tumor necrosis factor has been implicated in the pathophysiology ofvisceral pain (see Coelho et al. (2000) Am J Physiol Gastrointest LiverPhysiol. 279:G781; Coelho et al. (2000) Brain Res Bull. 52:223).Visceral pain is used to refer to nociceptive pain that is mediated byreceptors on A-delta and C nerve fibers. A-delta and C-nerve fibers arewhich are located in skin, bone, connective tissue, muscle and viscera.Visceral pain can be vague in distribution, spasmodic in nature and isusually described as deep, aching, squeezing and colicky in nature.Examples of visceral pain include pain associated with a heart attack,wherein the visceral pain can be felt in the arm, neck and/or back, andliver capsule pain, wherein the visceral pain can be felt in the backand/or right shoulder. Accordingly, the multiple-variable dose methodsof the invention can be used to treat visceral pain.

M. Hepatic Disorders

TNFα has been implicated in the pathophysiology of a wide variety ofhepatic disorders (see e.g., Colletti et al. (1990) J Clin Invest.85:1936; Tiegs (1997) Acta Gastroenterol Belg. 60:176; Fernandez et al.(2000) J Endotoxin Res. 6:321). The invention provides multiple-variabledose methods for inhibiting TNFα activity in a subject suffering fromsuch a hepatic disorder.

As used herein, the term “a hepatic disorder in which TNFα activity isdetrimental” is intended to include diseases and other disorders of theliver or conditions associated with hepatocellular injury or a biliarytract disorders in which the presence of TNFα in a subject sufferingfrom the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder. Accordingly, a hepaticdisorder in which TNFα activity is detrimental is a disorder in whichinhibition of TNFα activity is expected to alleviate the symptoms and/orprogression of the hepatic disorder. In one embodiment, hepaticdisorders refers to a human liver disease or condition associated withhepatocellular injury or a biliary tract disorder excluding hepatitis,alcoholic hepatitis, and viral hepatitis.

Examples of animal models used for evaluating the therapeutic efficacyof an agent for treating a hepatic disorder using multiple-variable dosemethods include the chimpanzee hepatitis C virus model (see Shimizu etal. (1990) Proc Natl Acad Sci. USA 87:6441). Examples of animal modelsused to study skin and nail disorder disorders include, for example, thesevere combined immunodeficient (SCID) mouse model (psoriasis) and theSmith line (SL) chicken and depigmenting mouse (vitiligo) (see Nickoloff(2000) Investig Dermatol Symp Proc. 5:67; Austin et al. (1995) Am JPathol. 146:1529; Lerner et al. (1986) J Invest Dermatol. 87:299).

Hepatic disorders include many diseases and disorders wherein the liverfunctions improperly or ceases to function. Hepatocellular injuries caninclude alcoholic cirrhosis, α1 antitypsin deficiency, autoimmunecirrhosis, cryptogenic cirrhosis, fulminant hepatitis, hepatitis B andC, and steatohepatitis. Examples of biliary tract disorders includecystic fibrosis, primary biliary cirrhosis, sclerosing cholangitis andbiliary obstruction (Wiesner (1996) “Current Indications, ContraIndications and Timing for Liver Transplantation” in Transplantation ofthe Liver, Saunders (publ.); Busuttil and Klintmalm (eds.) Chapter 6;Klein (1998) Partial Hypertension: The Role of Liver Transplantation,Musby (publ.) in Current Surgical Therapy 6.sup.th Ed. Cameron, J. (ed).

The term “hepatitis” refers to inflammation of the liver. Hepatitis canbe caused by infections with various organisms, including bacteria,viruses (Hepatitis A, B, C, etc.), or parasites. Chemical toxins such asalcohol, drugs, or poisonous mushrooms can also damage the liver andcause it to become inflamed. A rare but extremely dangerous cause ofhepatitis results from overdose of acetaminophen (Tylenol), which can bedeadly. In addition, immune cells in the body may attack the liver andcause autoimmune hepatitis. Hepatitis may resolve quickly (acutehepatitis), or cause long-term disease (chronic hepatitis). In someinstances, progressive liver damage or liver failure may result. Theincidence and severity of hepatitis vary depending on many factors,including the cause of the liver damage and any underlying illnesses ina patient.

In one embodiment, the invention features multiple-variable methods fortreating a hepatic disorder in which TNFα activity is detrimental,comprising administering to a subject an effective amount of a TNFαinhibitor in an induction dose and subsequently in a treatment dose,such that said disorder is treated. In one embodiment, the hepaticdisorder is selected from the group consisting of hepatitis C virus,autoimmune hepatitis, fatty-liver disease, hepatitis B virus,hepatotoxicity, and non-alcoholic hepatitis, including non-alcoholicsteatohepatitis (NASH). Examples of hepatic disorders are furtherdescribed below.

1. Hepatitis C Virus (HCV)

Tumor necrosis factor has been implicated in the pathophysiology of thehepatitis C virus (see Gonzalez-Amaro. (1994) J Exp Med. 179:841; Nelsonet al. (1997) Dig Dis Sci 42:2487; Kallinowski et al. (1998) Clin ExpImmunol. 111:269). The term “hepatitis C virus” or “HCV” is used todescribe the hepatitis virus which is the causative agent of non-A,non-B hepatitis. Hepatitis C virus causes an inflammation of the liver.HCV infection causes hepatitis C. Hepatitis C in the acute stage is, ingeneral, milder than hepatitis B, but a greater proportion of suchinfections become chronic. HCV is a major cause of acute hepatitis andchronic liver disease, including cirrhosis and liver cancer. HCV is oneof the viruses (A, B, C, D, and E), which together account for the vastmajority of cases of viral hepatitis. It is an enveloped RNA virus inthe flaviviridae family which appears to have a narrow host range. Animportant feature of the virus is the relative mutability of its genome,which in turn is probably related to the high propensity (80%) ofinducing chronic infection. HCV is clustered into several distinctgenotypes which may be important in determining the severity of thedisease and the response to treatment. In one embodiment, the inventionprovides a multiple-variable dose method for treating HCV.

2. Autoimmune Hepatitis (AIH)

Tumor necrosis factor has been implicated in the pathophysiology ofautoimmune hepatitis (see Cookson et al., (1999) Hepatology 30:851;Jazrawi et al., (2003) Liver Transpl. 9:377). As used herein,“autoimmune hepatitis” refers to a hepatic disorder characterized byinflammation of the liver caused by rogue immune cells that mistake theliver's normal cells for a foreign tissue or pathogen (disease-causingagent). Autoimmune hepatitis is often responsible for a progressivedestruction of the hepatic parenchyma with a high mortality if leftuntreated (Johnson et al. (1993) Hepatology, 18:998). One of thecharacteristics of autoimmune hepatitis is the presence of circulatingautoantibodies in almost 90% of patients' sera. Such antibodies can beused to identify subjects who have autoimmune hepatitis.

Clinical and serological differences between patients have lead to theclassification of AIH into two types. Type 1 is characterized by thepresence of anti-smooth muscle (SMA) and/or anti-nuclear antibodies(ANA) in patients' sera, while sera from Type II patients showanti-liver kidney microsomal antibodies type 1 (LKM1) (Homberg et al.,(1987) Hepatology, 7:1333; Maggiore et al. (1993) J. Pediatr.Gastroenterol Nutr. 17:376). A serological marker, anti-liver cytosoltype I antibodies (LC1), has been identified in 30% of patients with anAIH type II. In addition, LC1 proved to be the only serological markerin 10% of patients tested (Martini et al. (1988) Hepatology, 8:1662). Inone embodiment, the multiple-variable dose method of the invention isused to treat AIH.

3. Fatty-Liver Disease

Tumor necrosis factor has been implicated in the pathophysiology offatty-liver disease (see Valenti et al., (2002) Gastroenerology 122:274;Li et al., (2003) Hepatology 37:343). Fatty-liver disease refers to adisease wherein fat (hepatocytes) is excessively accumulated in theliver. Fatty liver disease is believed to be caused by supernutrition,hyperingestion of alcohol, diabetes and side effects due toadministration of pharmaceuticals. Fatty liver disease can cause severediseases such as chronic hepatitis and hepatic cirrhosis. In patientswith fatty liver disease, lipids, particularly neutral fat, accumulatein hepatocytes to the extent that the amount exceeds the physiologicallypermissible range. From a biochemical point of view, a standard forjudgment of fatty liver is that the weight of neutral fat is about 10%(100 mg/g wet weight) or more of the wet weight of hepatic tissue. Inone embodiment, the multiple-variable dose method of the invention isused to treat fatty liver disease.

4. Hepatitis B Virus (HBV)

Tumor necrosis factor has been implicated in the pathophysiology ofhepatitis B virus (see Kasahara et al., (2003) J Virol. 77:2469; Wang(2003) World J Gastroenterol. 9:641; Biermer et al. (2003) J Virol.77:4033). The term “hepatitis B virus” (HBV) is used to describe thevirus (serum hepatitis virus) which produces viral hepatitis type B inhumans. This is a viral disease with a long incubation period (about 50to 160 days) in contrast to hepatitis A virus (infectious hepatitisvirus) which has a short incubation period. The hepatitis B virus isusually transmitted by injection of infected blood or blood derivativesor merely by use of contaminated needles, lancets or other instruments.Clinically and pathologically, the disease is similar to viral hepatitistype A; however, there is no cross-protective immunity. Viral antigen(HBAg) is found in the serum after infection.

Hepatitis B virus infects humans at a very high rate. Most people whobecome infected with Hepatitis B get rid of the virus within 6 months,wherein a short infection is known as an “acute” case of Hepatitis B. Itis estimated that at least about 300 million people are chronic carriersof HBV. Infection with the virus results in a range of clinical symptomsincluding minor flu-like symptoms to death. In one embodiment, themultiple-variable dose method of the invention is used to treat HBVinfection.

5. Hepatotoxicity

Tumor necrosis factor has been implicated in the pathophysiology ofhepatotoxicity (see Bruccoleri et al. (1997) Hepatology 25:133; Lusteret al. (2000) Ann NY Acad Sci. 919:214; Simeonova et al. (2001) ToxicolAppl Pharmacol. 177:112). The term hepatotoxicity refers to liver damagecaused by medications and other chemicals or drugs. The best indicatorfor identifying liver toxicity in a subject is the elevation of certainenzyme measurements in the blood, such as AST (aspartateaminotransferase), ALT (alanine aminotransferase), and GOT (glutamateoxalacetate transaminase).

Hepatotoxicity can cause permanent injury and death. Initial symptoms ofhepatotoxicity can include acute gastrointestinal symptoms, e.g., severediarrhea. The second phase of hepatotoxicity is characterized byabatement of symptoms. During this apparent subsidence, biochemicalevidence of hepatic injury appears. Oliguria (decreased urine output) isusual during the second phase. The third phase, that of overt hepaticdamage, becomes clinically apparent 3 to 5 days after ingestion of thechemical, with the appearance of jaundice. Renal failure may also occur.The symptoms of chemically-induced (drug-induced) hepatitis are similarto that of infectious hepatitis. In one embodiment, themultiple-variable dose method of the invention is used to treathepatotoxicity.

6. Liver Failure (e.g. Chronic Liver Failure)

Tumor necrosis factor has been implicated in the pathophysiology ofliver failure (e.g. chronic liver failure) (see Takenaka et al., (1998)Dig Dis Sci. 43:887; Nagaki et al. (1999) J Hepatol. 31:997; Streetz etal., (2000) Gastroenterology. 119:446. Liver failure, including chronicliver failure, usually develops over a period of years and is caused bya repeated insult to the liver (such as alcohol abuse or infection withhepatitis virus) which slowly damages the organ. Less commonly, liverfailure is acute, and occurs over a period of days or weeks. Causes ofacute liver failure include hepatitis virus infections, drugs,pregnancy, autoimmune disease, and sudden low blood flow to the liver.In one embodiment, the multiple-variable dose method of the invention isused to treat liver failure.

7. Non-Alcoholic Hepatitis, Including NASH

Tumor necrosis factor has been implicated in the pathophysiology ofnon-alcoholic hepatitis, including nonalcoholic steatohepatitis (seeCrespo et al., (2001) Hepatology. 34:1158; Pessayre et al. (2002)282(2):G193). The term “nonalcoholic steatohepatitis” or “NASH” refersto the development of histologic changes in the liver that arecomparable to those induced by excessive alcohol intake, but in theabsence of alcohol abuse. NASH is characterized by macrovesicular and/ormicrovesicular steatosis, lobular and portal inflammation, andoccasionally Mallory bodies with fibrosis and cirrhosis. NASH is alsocommonly associated with hyperlipidemia, obesity, and type II diabetesmellitus.

Additional clinical conditions which characterize hepatic steatosis andinflammation include excessive fasting, jejunoileal bypass, totalparental nutrition, chronic hepatitis C, Wilson's disease, and adversedrug effects such as those from corticosteroids, calcium channelblockers, high dose synthetic estrogens, methotrexate and amiodarone.Thus, the term “nonalcoholic steatohepatitis” can be used to describethose patients who exhibit these biopsy findings, coupled with theabsence of (a) significant alcohol consumption, (b) previous surgery forweight loss, (c) history of drug use associated with steatohepatitis,(d) evidence of genetic liver disease or (e) chronic hepatitis Cinfection (see, e.g., Ludwig et al., (1980) Mayo Clin. Proc. 55:434;Powell et al. (1990) Hepatol. 11:74). In one embodiment, themultiple-variable dose method of the invention is used to treat NASH.

N. Skin and Nail Disorders

Tumor necrosis factor has been implicated in the pathophysiology of skinand nail disorders. In one embodiment, the multiple-variable dose methodof the invention comprising administering an induction dose of a TNFαantibody followed by a subsequent treatment dose, can be used to treatskin and nail disorders. The term “skin disorder” or “skin disease” asused interchangeably herein, refers to abnormalities, other than injurywounds, of the skin which have induced a state of inflammation. In oneembodiment, the skin disorder of the invention is an inflammatory skindisorder, wherein the skin is characterized by capillary dilatation,leukocytic infiltration, redness, heat, and/or pain. Examples of skindisorders include, but are not limited to, psoriasis, pemphigusvulgaris, scleroderma, atopic dermatitis, sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, and vitiligo.As used herein, the term “skin and nail disorder in which TNFα activityis detrimental” is intended to include skin and/or nail disorders andother disorders in which the presence of TNFα in a subject sufferingfrom the disorder has been shown to be or is suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder, e.g., psoriasis.Accordingly, skin and nail disorders in which TNFα activity isdetrimental are disorders in which inhibition of TNFα activity isexpected to alleviate the symptoms and/or progression of the disorder.The use of the antibodies, antibody portions, and other TNFα inhibitorsof the invention in the treatment of specific skin and nail disorders isdiscussed further below. In certain embodiments, the treatment method ofthe invention is performed in combination with another therapeuticagent, as described below in Section IV. In one embodiment, themultiple-variable dose method of the invention comprising administeringa TNFα antibody in combination with another therapeutic agent is usedfor the treatment of psoriasis and the treatment of psoriasis associatedwith arthritis.

1. Psoriasis

Tumor necrosis factor has been implicated in the pathophysiology ofpsoriasis (Takematsu et al. (1989) Arch Dermatol Res. 281:398; Victorand Gottlieb (2002) J Drugs Dermatol. 1:264). The term “psoriasis” asused herein, refers to skin disorders associated with epidermalhyperplasia. Example of psoriasis include, but are not limited to,chronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis, psoriasis vulgaris, and erythrodermic psoriasis. Psoriasiscan also be associated with other inflammatory disorders, includinginflammatory bowel disease (IBD) and rheumatoid arthritis (RA).

Psoriasis is described as a skin inflammation (irritation and redness)characterized by frequent episodes of redness, itching, and thick, dry,silvery scales on the skin. In particular, lesions are formed whichinvolve primary and secondary alterations in epidermal proliferation,inflammatory responses of the skin, and an expression of regulatorymolecules such as lymphokines and inflammatory factors. Psoriatic skinis morphologically characterized by an increased turnover of epidermalcells, thickened epidermis, abnormal keratinization, inflammatory cellinfiltrates into the epidermis and polymorphonuclear leukocyte andlymphocyte infiltration into the epidermis layer resulting in anincrease in the basal cell cycle. Psoriasis often involves the nails,which frequently exhibit pitting, separation of the nail, thickening,and discoloration. Psoriasis is often associated with other inflammatorydisorders, for example arthritis, including rheumatoid arthritis,inflammatory bowel disease (IBD), and Crohn's disease. Approximately onethrid of subjects with psoriasis also have psoriatic arthritis (PsA)which, as described above, causes stiffness, swelling of the joints,pain, and reducd range of motion (Greaves et al. (1995) N Eng. J. Med.332:581).

Evidence of psoriasis is most commonly seen on the trunk, elbows, knees,scalp, skin folds, or fingernails, but it may affect any or all parts ofthe skin. Normally, it takes about a month for new skin cells to move upfrom the lower layers to the surface. In psoriasis, this process takesonly a few days, resulting in a build-up of dead skin cells andformation of thick scales. Symptoms of psoriasis include: skin patches,that are dry or red, covered with silvery scales, raised patches ofskin, accompanied by red borders, that may crack and become painful, andthat are usually located on the elbows, knees, trunk, scalp, and hands;skin lesions, including pustules, cracking of the skin, and skinredness; joint pain or aching which may be associated with of arthritis,e.g., psoriatic arthritis.

Treatment for psoriasis often includes a topical corticosteroids,vitamin D analogs, and topical or oral retinoids, or combinationsthereof. In one embodiment, the TNFα inhibitor of the invention isadministered in combination with or the presence of one of these commontreatments. Additional therapeutic agents which can also be combinedwith the TNFα inhibitor of the invention for treatment of psoriasis aredescribed in more detail in Section IV.

The diagnosis of psoriasis is usually based on the appearance of theskin. Additionally a skin biopsy, or scraping and culture of skinpatches may be needed to rule out other skin disorders. An x-ray may beused to check for psoriatic arthritis if joint pain is present andpersistent.

Improvements in psoriasis in a subject can be monitored by the subject'sPsoriasis Area and Severity Index Score (PAST). The method fordetermining the PASI has been described in Fredriksson and Pettersson(1978) Dermatologica 157:238 and Marks et al. (1989) Arch Dermatol125:235. Briefly, the index is based on evaluation of four anatomicsites, including the head, upper extremities, trunk, and lowerextremities, for erythema, induration, and desquamation using a 5 pointscale (0=no symptoms; 1=slight; 2=moderate; 3=marked; 4=very marked).Based on the extent of lesions in a given anatomic site, the areaaffected is assigned a numerical value (0=0; 1=<10%; 2=10-29%; 3=30-49%;4=50-69%; 5=70=89%; 6=90-100%). The PASI score is then calculated,wherein the possible range of PASI score is 0.0 to 72.0 with the highestscore representing complete erythroderma of the severest degree.

In one embodiment of the invention, a TNFα inhibitor is used inmultiple-variable dose treatment for psoriasis, including chronic plaquepsoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis,pemphigus vulgaris, erythrodermic psoriasis, psoriasis associated withinflammatory bowel disease (IBD), and psoriasis associated withrheumatoid arthritis (RA). In another embodiment, a TNFα inhibitor, suchas D2E7, is used in a multiple variable dose regimen to treat subjectswho have psoriasis in combination with PsA. Specific types of psoriasisincluded in the treatment methods of the invention are described indetail below:

a. Chronic Plaque Psoriasis

Tumor necrosis factor has been implicated in the pathophysiology ofchronic plaque psoriasis (Asadullah et al. (1999) Br J Dermatol.141:94). Chronic plaque psoriasis (also referred to as psoriasisvulgaris) is the most common form of psoriasis. Chronic plaque psoriasisis characterized by raised reddened patches of skin, ranging fromcoin-sized to much larger. In chronic plaque psoriasis, the plaques maybe single or multiple, they may vary in size from a few millimeters toseveral centimeters. The plaques are usually red with a scaly surface,and reflect light when gently scratched, creating a “silvery” effect.Lesions (which are often symmetrical) from chronic plaque psoriasisoccur all over body, but with predilection for extensor surfaces,including the knees, elbows, lumbosacral regions, scalp, and nails.Occasionally chronic plaque psoriasis can occur on the penis, vulva andflexures, but scaling is usually absent. Diagnosis of patients withchronic plaque psoriasis is usually based on the clinical featuresdescribed above. In particular, the distribution, color and typicalsilvery scaling of the lesion in chronic plaque psoriasis arecharacteristic of chronic plaque psoriasis.

b. Guttate Psoriasis

Guttate psoriasis refers to a form of psoriasis with characteristicwater drop shaped scaly plaques. Flares of guttate psoriasis generallyfollow an infection, most notably a streptococcal throat infection.Diagnosis of guttate psoriasis is usually based on the appearance of theskin, and the fact that there is often a history of recent sore throat.

c. Inverse Psoriasis

Inverse psoriasis is a form of psoriasis in which the patient hassmooth, usually moist areas of skin that are red and inflammed, which isunlike the scaling associated with plaque psoriasis. Inverse psoriasisis also referred to as intertiginous psoriasis or flexural psoriasis.Inverse psoriasis occurs mostly in the armpits, groin, under the breastsand in other skin folds around the genitals and buttocks, and, as aresult of the locations of presentation, rubbing and sweating canirriate the affected areas.

d. Pustular Psoriasis

Pustular psoriasis, also referred to as palmar plantar psoriasis, is aform of psoriasis that causes pus-filled blisters that vary in size andlocation, but often occur on the hands and feet. The blisters may belocalized, or spread over large areas of the body. Pustular psoriasiscan be both tender and painful, can cause fevers.

e. Other Psoriasis Disorders

Other examples of psoriatic disorders which can be treated with the TNFαantibody of the invention include erythrodermic psoriasis, vulgaris,psoriasis associated with IBD, and psoriasis associated with arthritis,including rheumatoid arthritis.

2. Pemphigus vulgaris

Pemphigus vulgaris is a serious autoimmune systemic dermatologic diseasethat often affects the oral mucous membrane and skin. The pathogenesisof pemphigus vulgaris is thought to be an autoimmune process that isdirected at skin and oral mucous membrane desmosomes. Consequentially,cells do not adhere to each other. The disorder manifests as largefluid-filled, rupture-prone bullae, and has a distinctive histologicappearance. Anti-inflammatory agents are the only effective therapy forthis disease which has a high mortality rate. Complications that arisein patients suffering from pemphigus vulgaris are intractable pain,interference with nutrition and fluid loss, and infections.

3. Atopic Dermatitis/Eczema

Atopic dermatitis (also referred to as eczema) is a chronic skindisorder categorized by scaly and itching plaques. People with eczemaoften have a family history of allergic conditions like asthma, hayfever, or eczema. Atopic dermatitis is a hypersensitivity reaction(similar to an allergy) which occurs in the skin, causing chronicinflammation. The inflammation causes the skin to become itchy andscaly. Chronic irritation and scratching can cause the skin to thickenand become leathery-textured. Exposure to environmental irritants canworsen symptoms, as can dryness of the skin, exposure to water,temperature changes, and stress.

Subjects with atopic dermatitis can be identified by certain symptoms,which often include intense itching, blisters with oozing and crusting,skin redness or inflammation around the blisters, rash, dry, leatheryskin areas, raw areas of the skin from scratching, and eardischarges/bleeding.

4. Sarcoidosis

Sarcoidosis is a disease in which granulomatous inflammation occurs inthe lymph nodes, lungs, liver, eyes, skin, and/or other tissues.Sarcoidosis includes cutaneous sarcoidosis (sarcoidosis of the skin) andnodular sarcoidosis (sarcoidosis of the lymph nodes). Patients withsarcoidosis can be identified by the symptoms, which often includegeneral discomfort, uneasiness, or an ill feeling; fever; skin lesions.

5. Erythema Nodosum

Erythema nodosum refers to an inflammatory disorder that ischaracterized by tender, red nodules under the skin, typically on theanterior lower legs. Lesions associated with erythema nodosum oftenbegin as flat, but firm, hot red painful lumps (approximately an inchacross). Within a few days the lesions may become purplish, and thenover several weeks fade to a brownish flat patch.

In some instances, erythema nodosum may be associated with infectionsincluding, streptococcus, coccidioidomycosis, tuberculosis, hepatitis B,syphilis, cat scratch disease, tularemia, yersinia, leptospirosispsittacosis, histoplasmosis, mononucleosis (EBV). In other instances,erythema nodosum may be associated with sensitivity to certainmedications including, oralcontraceptives, penicillin, sulfonamides,sulfones, barbiturates, hydantoin, phenacetin, salicylates, iodides, andprogestin. Erythema nodosum is often associated with other disordersincluding, leukemia, sarcoidosis, rheumatic fever, and ulcerativecolitis.

Symptoms of erythema nodosum usually present themselves on the shins,but lesions may also occur on other areas of the body, including thebuttocks, calves, ankles, thighs and upper extremities. Other symptomsin subjects with erythema nodosum can include fever and malaise.

6. Hidradenitis Suppurative

Hidradenitis suppurativa refers to a skin disorder in which swollen,painful, inflamed lesions or lumps develop in the groin and sometimesunder the arms and under the breasts. Hidradenitis suppurativa occurswhen apocrine gland outlets become blocked by perspiration or are unableto drain normally because of incomplete gland development. Secretionstrapped in the glands force perspiration and bacteria into surroundingtissue, causing subcutaneous induration, inflammation, and infection.Hidradenitis suppurativa is confined to areas of the body that containapocrine glands. These areas are the axillae, areola of the nipple,groin, perineum, circumanal, and periumbilical regions.

7. Lichen Planus

Tumor necrosis factor has been implicated in the pathophysiology oflichen planus (Sklavounou et al. (2000) J Oral Pathol Med. 29:370).Lichen planus refers to a disorder of the skin and the mucous membranesresulting in inflammation, itching, and distinctive skin lesions. Lichenplanus may be associated with hepatitis C or certain medications.

8. Sweet's Syndrome

Inflammatory cytokines, including tumor necrosis factor, have beenimplicated in the pathophysiology of Sweet's syndrome (Reuss-Borst etal. (1993) Br J Haematol. 84:356). Sweet's syndrome, which was describedby R. D. Sweet in 1964, is characterized by the sudden onset of fever,leukocytosis, and cutaneous eruption. The eruption consists of tender,erythematous, well-demarcated papules and plaques which show denseneutrophilic infiltrates microscopically. The lesions may appearanywhere, but favor the upper body including the face. The individuallesions are often described as pseudovesicular or pseudopustular, butmay be frankly pustular, bullous, or ulcerative. Oral and eyeinvolvement (conjunctivitis or episcleritis) have also been frequentlyreported in patients with Sweet's syndrome. Leukemia has also beenassociated with Sweet's syndrome.

9. Vitiligo

Vitiligo refers to a skin condition in which there is loss of pigmentfrom areas of skin resulting in irregular white patches with normal skintexture. Lesions characteristic of vitiligo appear as flat depigmentedareas. The edges of the lesions are sharply defined but irregular.Frequently affected areas in subjects with vitiligo include the face,elbows and knees, hands and feet, and genitalia.

10. Scleroderma

Tumor necrosis factor has been implicated in the pathophysiology ofscleroderma (Tutuncu et al. (2002) Clin Exp Rheumatol. 20(6 Suppl28):S146; Mackiewicz et al. (2003) Clin Exp Rheumatol. 21:41; Murota etal. (2003) Arthritis Rheum. 48:1117). Scleroderma refers to a a diffuseconnective tissue disease characterized by changes in the skin, bloodvessels, skeletal muscles, and internal organs. Scleroderma is alsoreferred to as CREST syndrome or progressive systemic sclerosis, andusually affects people between the ages 30-50. Women are affected moreoften than men.

The cause of scleroderma is unknown. The disease may produce local orsystemic symptoms. The course and severity of the disease varies widelyin those affected. Excess collagen deposits in the skin and other organsproduce the symptoms. Damage to small blood vessels within the skin andaffected organs also occurs. In the skin, ulceration, calcification, andchanges in pigmentation may occur. Systemic features may includefibrosis and degeneration of the heart, lungs, kidneys andgastrointestinal tract.

Patients suffering from scleroderma exhibit certain clinical features,including, blanching, blueness, or redness of fingers and toes inresponse to heat and cold (Raynaud's phenomenon), pain, stiffness, andswelling of fingers and joints, skin thickening and shiny hands andforearm, esophageal reflux or heartburn, difficulty swallowing, andshortness of breath. Other clinical sypmtoms used to diagnosescleroderma include, an elevated erythrocyte sedimentaion rate (ESR), anelevated rheumatoid factor (RF), a positive antinuclear antibody test,urinalysis that shows protein and microscopic blood, a chest X-ray thatmay show fibrosis, and pulmonary funtion studies that show restricitivelung disease.

11. Nail Disorders

Nail disorders include any abnormality of the nail. The term “naildisorder” or “nail disease” as used herein, refers to conditions whereinthe fingernails or toenails to abnormal color, shape, texture, orthickness. Specific nail disorders include, but are not limited to,pitting, koilonychia, Beau's lines, spoon nails, onycholysis, yellownails, pterygium (seen in lichen planus), and leukonychia. Pitting ischaracterised by the presence of small depressions on the nail surface.Ridges or linear elevations can develop along the nail occurring in a“lengthwise” or “crosswise” direction. Beau's lines are lineardepressions that occur “crosswise” (transverse) in the fingernail.Leukonychia describes white streaks or spots on the nails. Koilonychiais an abnormal shape of the fingernail where the nail has raised ridgesand is thin and concave Koilonychia is often associated with irondeficiency.

Nail disorders which can be treated with the TNFα antibody of theinvention also include psoriatic nails. Psoriatic nails include changesin nails which are attributable to psoriasis. In some instancespsoriasis may occur only in the nails and nowhere else on the body.Psoriatic changes in nails range from mild to severe, generallyreflecting the extent of psoriatic involvement of the nail plate, nailmatrix, i.e., tissue from which the nail grows, nail bed, i.e., tissueunder the nail, and skin at the base of the nail. Damage to the nail bedby the pustular type of psoriasis can result in loss of the nail. Nailchanges in psoriasis fall into general categories that may occur singlyor all together. In one category of psoriatic nails, the nail plate isdeeply pitted, probably due to defects in nail growth caused bypsoriasis. In another category, the nail has a yellow to yellow-pinkdiscoloration, probably due to psoriatic involvement of the nail bed. Athird subtype of psoriatic nails are characterized by white areas whichappear under the nail plate. The white areas are actually air bubblesmarking spots where the nail plate is becoming detached from the nailbed. There may also be reddened skin around the nail. A fourth categoryis evidenced by the nail plate crumbling in yellowish patches, i.e.,onychodystrophy, probably due to psoriatic involvement in the nailmatrix. A fifth category is characterized by the loss of the nail in itsentirety due to psoriatic involvement of the nail matrix and nail bed.

The multiple-variable dose method of treatment of the invention can alsobe used to treat nail disorders often associated with lichen planus.Nails in subjects with lichen planus often show thinning and surfaceroughness of the nail plate with longitudinal ridges or pterygium.

The multiple-variable dose method of treatment of the invention can beused to treat nail disorders, such as those described herein. Often naildisorders are associated with skin disorders. In one embodiment, theinvention includes a multiple-variable dose method of treatment for naildisorders using a TNFα antibody. In another embodiment, the naildisorder is associated with another disorder, including a skin disordersuch as psoriasis. In another embodiment, the disorder associated with anail disorder is arthritis, including psoriatic arthritis.

12. Other Skin and Nail Disorders

The multiple-variable dose method of treatment of the invention can beused to treat other skin and nail disorders, such as chronic actinicdermatitis, bullous pemphigoid, and alopecia areata. Chronic actinicdermatitis (CAD) is also referred to as photosensitivitydermatitis/actinic reticuloid syndrome (PD/AR). CAD is a condition inwhich the skin becomes inflamed, particularly in areas that have beenexposed to sunlight or artificial light. Commonly, CAD patients haveallergies to certain substances that come into contact with their skin,particularly various flowers, woods, perfumes, sunscreens and rubbercompounds. Bullous pemphigoid refers to a skin disorder characterized bythe formation of large blisters on the trunk and extremities. Alopeciaareata refers to hair loss characterized by round patches of completebaldness in the scalp or beard.

O. Vasculitides

TNFα has been implicated in the pathophysiology of a variety ofvasculitides, (see e.g., Deguchi et al. (1989) Lancet. 2:745). In oneembodiment, the invention provides a multiple-variable dose method forinhibiting TNFα activity in a subject suffering from a vasculitis inwhich TNFα activity is detrimental.

The term “vasculitis” or “vasculitides” as used interchangeably herein,refers to a group of disorders which are characterized by theinflammation of blood vessels. Blood vessels of all sizes may beaffected, from the largest vessel in the body (the aorta) to thesmallest blood vessels in the skin (capillaries). The size of bloodvessel affected varies according to the specific type of vasculitis. Asused herein, the term “a vasculitis in which TNFα activity isdetrimental” is intended to include vasculitis in which the presence ofTNFα in a subject suffering from the disorder has been shown to be or issuspected of being either responsible for the pathophysiology of thedisorder or a factor that contributes to a worsening of the disorder.Such disorders may be evidenced, for example, by an increase in theconcentration of TNFα in a biological fluid of a subject suffering fromthe disorder (e.g., an increase in the concentration of TNFα in serum,plasma, synovial fluid, etc. of the subject), which can be detected, forexample, using an anti-TNFα antibody as described above.

There are numerous examples of vasculitides in which TNFα activity isdetrimental, including Behcet's disease. The use of the antibodies,antibody portions, and other TNFα inhibitors for multiple-variable dosetreatment of the invention of specific vasculitides is discussed furtherbelow. In certain embodiments, the antibody, antibody portion, or otherTNFα inhibitor of the invention is administered to the subject incombination with another therapeutic agent, as described below

The multiple-variable dose regimen of the invention can be used to treatvasculitis in which TNFα activity is detrimental, wherein inhibition ofTNFα activity is expected to alleviate the symptoms and/or progressionof the vasculitis or to prevent the vasculitis. Subjects suffering fromor at risk of developing vasculitis can be identified through clinicalsymptoms and tests. For example, subjects with vasculitides oftendevelop antibodies to certain proteins in the cytoplasm of neutrophils,antineutrophil cytoplasmic antibodies (ANCA). Thus, in some instances,vasculitides may be evidenced by tests (e.g., ELISA), which measure ANCApresence.

Vasculitis and its consequences may be the sole manifestation of diseaseor it may be a secondary component of another primary disease.Vasculitis may be confined to a single organ or it may simultaneouslyaffect several organs. and depending on the syndrome, arteries and veinsof all sizes can be affected. Vasculitis can affect any organ in thebody.

In vasculitis, the vessel lumen is usually compromised, which isassociated with ischemia of the tissues supplied by the involved vessel.The broad range of disorders that may result from this process is due tothe fact that any type, size and location of vessel (e.g., artery, vein,arteriole, venule, capillary) can be involved. Vasculitides aregenerally classified according to the size of the affected vessels, asdescribed below. It should be noted that some small and large vesselvasculitides may involve medium-sized arteries; but large andmedium-sized vessel vasculitides do not involve vessels smaller thanarteries. Large vessel disease includes, but is not limited to, giantcell arteritis, also known as temporal arteritis or cranial arteritis,polymyalgia rheumatica, and Takayasu's disease or arteritis, which isalso known as aortic arch syndrome, young female arteritis and Pulselessdisease. Medium vessel disease includes, but is not limited to, classicpolyarteritis nodosa and Kawasaki's disease, also known as mucocutaneouslymph node syndrome. Non-limiting examples of small vessel disease areBehcet's Syndrome, Wegner's granulomatosis, microscopic polyangitis,hypersensitivity vasculitis, also known as cutaneous vasculitis, smallvessel vasculitis, Henoch-Schonlein purpura, allergic granulamotosis andvasculitis, also known as Churg Strauss syndrome. Other vasculitidesinclude, but are not limited to, isolated central nervous systemvasculitis, and thromboangitis obliterans, also known as Buerger'sdisease. Classic Polyarteritis nodosa (PAN), microscopic PAN, andallergic granulomatosis are also often grouped together and are calledthe systemic necrotizing vasculitides. A further description ofvasculitis is described below:

1. Large Vessel Vasculitis

In one embodiment, the TNFα antibody of the invention is used to treatsubjects who have large vessel vasculitis. The term “large vessel(s)” asused herein, refers to the aorta and the largest branches directedtoward major body regions. Large vessels include, for example, theaorta, and its branches and corresponding veins, e.g., the subclavianartery; the brachiocephalic artery; the common carotid artery; theinnonimate vein; internal and external jugular veins; the pulmonaryarteries and veins; the venae cavae; the renal arteries and veins; thefemoral arteries and veins; and the carotid arteries. Examples of largevessel vasculitides are described below.

a. Giant Cell Arteritis (GCA)

Tumor necrosis factor has been implicated in the pathophysiology ofgiant cell arteritis (Sneller (2002) Cleve. Clin. J. Med. 69:SII40;Schett et al. (2002) Ann. Rheum. Dis. 61:463). Giant cell arteritis(GCA), refers to a vasculitis involving inflammation and damage to bloodvessels, particularly the large or medium arteries that branch from theexternal carotid artery of the neck. GCA is also referred to as temporalarteritis or cranial arteritis, and is the most common primaryvasculitis in the elderly. It almost exclusively affects individualsover 50 years of age, however, there are well-documented cases ofpatients 40 years and younger. GCA usually affects extracranialarteries. GCA can affect the branches of the carotid arteries, includingthe temporal artery. GCA is also a systemic disease which can involvearteries in multiple locations.

Histopathologically, GCA is a panarteritis with inflammatory mononuclearcell infiltrates within the vessel wall with frequent Langhans typegiant cell formation. There is proliferation of the intima,granulomatous inflammation and fragmentation of the internal elasticlamina. The pathological findings in organs is the result of ischemiarelated to the involved vessels.

Patients suffering from GCA exhibit certain clinical symptoms, includingfever, headache, anemia and high erythrocyte sedimentation rate (ESR).Other typical indications of GCA include jaw or tongue claudication,scalp tenderness, constitutional symptoms, pale optic disc edema(particularly ‘chalky white’ disc edema), and vision disturbances. Thediagnosis is confirmed by temporal artery biopsy.

b. Polymyalgia Rheumatica

Tumor necrosis factor has been implicated in the pathophysiology ofpolymyalgia rheumatica (Straub et al. (2002) Rheumatology (Oxford)41:423; Uddhammar et al. (1998) Br. J. Rheumatol. 37:766). Polymyalgiarheumatica refers to a rheumatic disorder that is associated withmoderate to severe muscle pain and stiffness in the neck, shoulder, andhip, most noticeable in the morning. IL-6 and IL-1β expression has alsobeen detected in a majority of the circulating monocytes in patientswith the polymyalgia rheumatica. Polymyalgia rheumatica may occurindependently, or it may coexist with or precede GCA, which is aninflammation of blood vessels.

c. Takayasu's Arteritis

Tumor necrosis factor has been implicated in the pathophysiology ofTakayasu's arteritis (Kobayashi and Numano (2002) Intern. Med. 41:44;Fraga and Medina (2002) Curr. Rheumatol. Rep. 4:30). Takayasu'sarteritis refers to a vasculitis characterized by an inflammation of theaorta and its major branches. Takayasu's arteritis (also known as Aorticarch syndrome, young female arteritis and Pulseless disease) affects thethoracic and abdominal aorta and its main branches or the pulmonaryarteries. Fibrotic thickening of the aortic wall and its branches (e.g.,carotid, inominate, and subclavian arteries) can lead to reduction oflumen size of vessels that arise from the aortic arch. This conditionalso typically affects the renal arteries.

Takayasu's arteritis primarily affects young women, usually aged 20-40years old, particularly of Asian descent, and may be manifested bymalaise, arthralgias and the gradual onset of extremity claudication.Most patients have asymmetrically reduced pulses, usually along with ablood pressure differential in the arms. Coronary and/or renal arterystenosis may occur.

The clinical features of Takayasu's arteritis may be divided into thefeatures of the early inflammatory disease and the features of the laterdisease. The clinical features of the early inflammatory stage ofTakayasu's disease are: malaise, low grade fever, weight loss, myalgia,arthralgia, and erythema multiforme. Later stages of Takayasu's diseaseare characterized by fibrotic stenosis of arteries and thrombosis. Themain resulting clinical features are ischaemic phenomena, e.g. weak andasymmetrical arterial pulses, blood pressure discrepancy between thearms, visual disturbance, e.g. scotomata and hemianopia, otherneurological features including vertigo and syncope, hemiparesis orstroke. The clinical features result from ischaemia due to arterialstenosis and thrombosis.

2. Medium Vessel Disease

In one embodiment, the TNFα antibody of the invention is used to treatsubjects who have medium vessel vasculitis. The term “medium vessel(s)”is used to refer to those blood vessels which are the main visceralarteries. Examples of medium vessels include the mesenteric arteries andveins, the iliac arteries and veins, and the maxillary arteries andveins. Examples of medium vessel vasculitides are described below.

a. Polyarteritis Nodosa

Tumor necrosis factor has been implicated in the pathophysiology ofpolyarteritis nodosa (DiGirolamo et al. (1997) J. Leukoc. Biol. 61:667).Polyarteritis nodosa, or periarteritis nodosa refers to vasculitis whichis a serious blood vessel disease in which small and medium-sizedarteries become swollen and damaged because they are attacked by rogueimmune cells. Polyarteritis nodosa usually affects adults morefrequently than children. It damages the tissues supplied by theaffected arteries because they don't receive enough oxygen andnourishment without a proper blood supply.

Symptoms which are exhibited in patients with polyarteritis nodosagenerally result from damage to affected organs, often the skin, heart,kidneys, and nervous system. Generalized symptoms of polyarteritisnodosa include fever, fatigue, weakness, loss of appetite, and weightloss. Muscle aches (myalgia) and joint aches (arthralgia) are common.The skin of subjects with polyarteritis nodosa may also show rashes,swelling, ulcers, and lumps (nodular lesions).

Classic PAN (polyarteritis nodosa) is a systemic arteritis of small tomedium muscular arteritis in which involvement of renal and visceralarteries is common Abdominal vessels have aneurysms or occlusions in 50%of PAN patients. Classic PAN does not involve the pulmonary arteriesalthough the bronchial vessels may be involved. Granulomas, significanteosinophilia and an allergic diathesis are not part of the syndrome.Although any organ system may be involved, the most commonmanifestations include peripheral neuropathy, mononeuritis multiplex,intestinal ischemia, renal ischemia, testicular pain and livedoreticularis.

b. Kawasaki's Disease

Tumor necrosis factor has been implicated in the pathophysiology ofKawasaki's disease (Sundel (2002) Curr. Rheumatol. Rep. 4:474; Gedalia(2002) Curr. Rheumatol. Rep. 4:25). Although the cause of Kawasaki'sdisease is unknown, it is associated with acute inflammation of thecoronary arteries, suggesting that the tissue damage associated withthis disease may be mediated by proinflammatory agents such as TNFα.Kawasaki's disease refers to a vasculitis that affects the mucusmembranes, lymph nodes, lining of the blood vessels, and the heart.Kawasaki's disease is also often referred to as mucocutaneous lymph nodesyndrome, mucocutaneous lymph node disease, and infantile polyarteritis.Subjects afflicted with Kawasaki's disease develop vasculitis ofteninvolving the coronary arteries which can lead to myocarditis andpericarditis. Often as the acute inflammation diminishes, the coronaryarteries may develop aneurysm, thrombosis, and lead to myocardialinfarction.

Kawasaki's disease is a febrile systemic vasculitis associated withedema in the palms and the soles of the feet, with enlargement ofcervical lymph nodes, cracked lips and “strawberry tongue”. Although theinflammatory response is found in vessels throughout the body, the mostcommon site of end-organ damage is the coronary arteries. Kawasaki'sDisease predominantly affects children under the age of 5. The highestincidence is in Japan but is becoming increasingly recognized in theWest and is now the leading cause of acquired heart disease in USchildren. The most serious complication of Kawasaki disease is coronaryarteritis and aneurysm formation that occurs in a third of untreatedpatients.

3. Small Vessel Disease

In one embodiment, the TNFα antibody of the invention is used to treatsubjects who have small vessel vasculitis. The term “small vessel(s)” isused to refer to arterioles, venules and capillaries. Arterioles arearteries that contain only 1 or 2 layers of sooth muscle cells and areterminal to and continuous with the capillary network. Venules carryblood from the capillary network to veins and capillaries connectarterioles and venules. Examples of small vessel vasculitides aredescribed below.

a. Behcet's Disease

Tumor necrosis factor has been implicated in the pathophysiology ofBehcet's disease (Sfikakis (2002) Ann. Rheum. Dis. 61:ii51-3; Dogan andFarah (2002) Oftalmologia. 52:23). Behcet's disease is a chronicdisorder that involves inflammation of blood vessels throughout thebody. Behcet's disease may also cause various types of skin lesions,arthritis, bowel inflammation, and meningitis (inflammation of themembranes of the brain and spinal cord). As a result of Behcet'sdisease, the subject with the disorder may have inflammation in tissuesand organs throughout the body, including the gastrointestinal tract,central nervous system, vascular system, lungs, and kidneys. Behcet'sdisease is three times more common in males than females and is morecommon in the eastern Mediterranean and Japan.

Subjects who have Behcet's disease may show clinical symptoms includingrecurrent oral ulcers (resembling canker sores), recurrent genitalulcers, and eye inflammation. Serum levels of TNFα, IL-8, IL-1, IL-6INF-γ and IL-12 are elevated in Behcet's patients, and the production ofthese factors has been shown to be elevated in the monocytes of Behcet'spatients (see, e.g., Inflammatory Disease of Blood Vessels (2001) MarcelDekker, Inc., eds. G. S. Hoffman and C. M. Weyand, p. 473).

b. Wegener's Granulomatosis

Tumor necrosis factor has been implicated in the pathophysiology ofWegener's granulomatosis (Marquez et al. (2003) Curr. Rheumatol. Rep.5:128; Harman and Margo (1998) Surv. Ophthalmol. 42:458). Wegener'sgranulomatosis refers to a vasculitis that causes inflammation of bloodvessels in the upper respiratory tract (nose, sinuses, ears), lungs, andkidneys. Wegener's granulomatosis is also referred to as midlinegranulomatosis. Wegener's granulomatosis includes a granulomatousinflammation involving the respiratory tract, and necrotizing vasculitisaffecting small to medium-sized vessels. Subjects who have Wegener'sgranulomatosis often also have arthritis (joint inflammation).Glomerulonephritis may also be present in affected subjects, butvirtually any organ may be involved.

Patients affected with Wegener's granulomatosis typically show clinicalsymptoms comprising recurrent sinusitis or epistaxis, mucosalulcerations, otitis media, cough, hemoptysis and dyspnea. The firstsymptoms of Wegener's granulomatosis frequently include upperrespiratory tract symptoms, joint pains, weakness, and tiredness.

c. Churg-Strauss Syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofChurg-Strauss syndrome (Gross (2002) Curr. Opin. Rheumatol. 14:11; Churg(2001) Mod. Pathol. 14:1284). Churg-Strauss syndrome refers to avasculitis that is systemic and shows early manifestation signs ofasthma and eosinophilia. Churg-Strauss syndrome is also referred to asallergic granulomatosis and angiitis, and occurs in the setting ofallergic rhinitis, asthma and eosinophilia. Sinusitis and pulmonaryinfiltrates also occur in Churg-Strauss syndrome, primarily affectingthe lung and heart. Peripheral neuropathy, coronary arteritis andgastrointestinal involvement are common.

Patients afflicted with Churg-Strauss syndrome can be diagnosedaccording to criteria established by the American College ofRheumatology (ACR). These criteria were intended to distinguish CSS fromother forms of vasculitis. Not all patients meet every criterion. Some,in fact, may have only 2 or 3 criteria, yet they are still classified asChurg-Strauss syndrome. The ACR selected 6 disease features (criteria)as being those that best distinguished Churg-Strauss syndrome from othervasculitides. These criteria include: 1) asthma; 2) eosinophilia [>10%on differential WBC count]; 3) mononeuropathy; 4) transient pulmonaryinfiltrates on chest X-rays; 5) paranasal sinus abnormalities; and 6)biopsy containing a blood vessel with extravascular eosinophils.

P. Other TNFα-Related Disorders

In one embodiment, the invention features a multiple-variable dosemethod for treating a TNFα-related disorder in which TNFα activity isdetrimental, comprising administering to a subject an induction dose ofa TNFα inhibitor and a subsequent treatment dose, such that saidTNFα-related disorder is treated. Examples of TNFα-related disorders inwhich TNFα activity is detrimental, are discussed further below.

1. Juvenile Arthritis

Tumor necrosis factor has been implicated in the pathophysiology ofjuvenile arthritis, including juvenile rheumatoid arthritis (Grom et al.(1996) Arthritis Rheum. 39:1703; Mangge et al. (1995) Arthritis Rheum.8:211). In one embodiment, the TNFα antibody of the invention is used totreat juvenile rheumatoid arthritis.

The term “juvenile rheumatoid arthritis” or “JRA” as used herein refersto a chronic, inflammatory disease which occurs before age 16 that maycause joint or connective tissue damage. JRA is also referred to asjuvenile chronic polyarthritis and Still's disease.

JRA causes joint inflammation and stiffness for more than 6 weeks in achild of 16 years of age or less. Inflammation causes redness, swelling,warmth, and soreness in the joints. Any joint can be affected andinflammation may limit the mobility of affected joints. One type of JRAcan also affect the internal organs.

JRA is often classified into three types by the number of jointsinvolved, the symptoms, and the presence or absence of certainantibodies found by a blood test. These classifications help thephysician determine how the disease will progress and whether theinternal organs or skin is affected. The classifications of JRA includethe following

a. Pauciarticular JRA, wherein the patient has four or fewer joints areaffected. Pauciarticular is the most common form of JRA, and typicallyaffects large joints, such as the knees.

b. Polyarticular HRA, wherein five or more joints are affected. Thesmall joints, such as those in the hands and feet, are most commonlyinvolved, but the disease may also affect large joints.

c. Systemic JRA is characterized by joint swelling, fever, a light skinrash, and may also affect internal organs such as the heart, liver,spleen, and lymph nodes. Systemic JRA is also referred to as it Still'sdisease. A small percentage of these children develop arthritis in manyjoints and can have severe arthritis that continues into adulthood.

2. Endometriosis

Tumor necrosis factor has been implicated in the pathophysiology ofendometriosis, as women with endometriosis have elevated peritoneallevels of TNF (Eisermann et al. (1988) Fertil Steril 50:573; Halme(1989) Am J Obstet Gynecol 161:1718; Mori et al. (1991)Am J ReprodImmunol 26:62; Taketani et al. (1992) Am J Obstet Gynecol 167:265;Overton et al. (1996) Hum Reprod 1996; 11:380). In one embodiment, theTNFα antibody of the invention is used to treat endometriosis. The term“endometriosis” as used herein refers to a condition in which the tissuethat normally lines the uterus (endometrium) grows in other areas of thebody, causing pain, irregular bleeding, and frequently infertility.

3. Prostatitis

Tumor necrosis factor has been implicated in the pathophysiology ofprostatitis, as men with chronic prostatitis and chronic pelvic painhave significantly higher levels of TNF and IL-1 in semen compared tocontrols (Alexander et al. (1998) Urology 52:744; Nadler et al. (2000) JUrol 164:214; Orhan et al. (2001) Int J Urol 8:495) Furthermore, in arat model of prostatitis TNF levels were also increased in comparison tocontrols (Asakawa et al. (2001) Hinyokika Kiyo 47:459; Harris et al.(2000) Prostate 44:25). In one embodiment, the TNFα antibody of theinvention is used to treat prostatitis.

The term “prostatitis” as used herein refers to an inflammation of theprostate. Prostatitis is also referred to as pelvic pain syndrome.Prostatitis manifests itself in a variety of forms, includingnonbacterial prostatitis, acute prostatitis, bacterial prostatitis, andacute prostatitis. Acute prostatitis refers to an inflammation of theprostate gland that develops suddenly. Acute prostatitis is usuallycaused by a bacterial infection of the prostate gland. Chronicprostatitis is an inflammation of the prostate gland that developsgradually, continues for a prolonged period, and typically has subtlesymptoms. Chronic prostatitis is also usually caused by a bacterialinfection

4. Choroidal Neovascularization

Tumor necrosis factor has been implicated in the pathophysiology ofchoroidal neovascularization. For example, in surgically excisedchoroidal neovascular membranes, neovascular vessels stained positivefor both TNF and IL-1 (Oh H et al. (1999) Invest Ophthalmol Vis Sci40:1891). In one embodiment, the TNFα antibody of the invention is usedto treat choroidal neovascularization. The term “choroidalneovascularization” as used herein refers to the growth of new bloodvessels that originate from the choroid through a break in the Bruchmembrane into the sub-retinal pigment epithelium (sub-RPE) or subretinalspace. Choroidal neovascularization (CNV) is a major cause of visualloss in patients with the condition.

5. Sciatica

Tumor necrosis factor has been implicated in the pathophysiology ofsciatica (Ozaktay et al. (2002) Eur Spine J. 11:467; Brisby et al.(2002) Eur Spine J. 11:62). In one embodiment, the TNFα antibody of theinvention is used to treat sciatica. The term “sciatica” as used hereinrefers to a condition involving impaired movement and/or sensation inthe leg, caused by damage to the sciatic nerve. Sciatica is alsocommonly referred to as neuropathy of the sciatic nerve and sciaticnerve dysfunction. Sciatica is a form of peripheral neuropathy. Itoccurs when there is damage to the sciatic nerve, located in the back ofthe leg. The sciatic nerve controls the muscles of the back of the kneeand lower leg and provides sensation to the back of the thigh, part ofthe lower leg and the sole of the foot. Sciatica can be indicative ofanother disorder, including a lumbar herniated disc, spinal stenosis,degenerative disc disease, isthmic spondyloisthesis and piniformissyndrome.

6. Sjogren's Syndrome

Tumor necrosis factor has been implicated in the pathophysiology ofSjogren's syndrome (Koski et al. (2001) Clin Exp Rheumatol. 19:131). Inone embodiment, the TNFα antibody of the invention is used to treatSjogren's syndrome. The term “Sjogren's syndrome” as used herein refersto a systemic inflammatory disorder characterized by dry mouth,decreased tearing, and other dry mucous membranes, and is oftenassociated with autoimmune rheumatic disorders, such as rheumatoidarthritis. Dryness of the eyes and mouth are the most common symptoms ofthis syndrome. The symptoms may occur alone, or with symptoms associatedwith rheumatoid arthritis or other connective tissue diseases. There maybe an associated enlargement of the salivary glands. Other organs maybecome affected. The syndrome may be associated with rheumatoidarthritis, systemic lupus erythematosus, scleroderma, polymyositis, andother diseases.

7. Uveitis

Tumor necrosis factor has been implicated in the pathophysiology ofuveitis (Wakefield and Lloyd (1992) Cytokine 4:1; Woon et al. (1998)Curr Eye Res. 17:955). In one embodiment, the TNFα antibody of theinvention is used to treat uveitis. The term “uveitis” as used hereinrefers to an inflammation of the the uvea, which is the layer betweenthe sclera and the retina, which includes the iris, ciliary body, andthe choroid. Uveitis is also commonly referred to as iritis, parsplanitis, chroiditis, chorioretinitis, anterior uveitis, and posterioruveitis. The most common form of uveitis is anterior uveitis, whichinvolves inflammation in the front part of the eye, which is usuallyisolated to the iris. This condition is often called iritis. In oneembodiment, the term uveitis refers to an inflammation of the the uveawhich excludes inflammation associated with an autoimmune disease, i.e.,excludes autoimmune uveitis.

8. Wet Macular Degeneration

Tumor necrosis factor has been implicated in the pathophysiology of wetmacular degeneration. In one embodiment, the TNFα antibody of theinvention is used to treat wet macular degeneration. The term “wetmacular degeneration” as used herein refers to a disorder that affectsthe macula (the central part of the retina of the eye) and causesdecreased visual acuity and possible loss of central vision. Patientswith wet macular degeneration develop new blood vessels under theretina, which causes hemorrhage, swelling, and scar tissue.

9. Osteoporosis

Tumor necrosis factor has been implicated in the pathophysiology ofosteoporosis, (Tsutsumimoto et al. (1999) J Bone Miner Res. 14:1751).Osteoporosis is used to refer to a disorder characterized by theprogressive loss of bone density and thinning of bone tissue.Osteoporosis occurs when the body fails to form enough new bone, or whentoo much old bone is reabsorbed by the body, or both. The TNFα antibody,or antigen-binding fragment thereof, of the invention can be used totreat osteoporosis.

10. Osteoarthritis

Tumor necrosis factor has been implicated in the pathophysiology ofosteoarthritis, (Venn et al. (1993) Arthritis Rheum. 36:819; Westacottet al. (1994) J Rheumatol. 21:1710). Osteoarthritis (OA) is alsoreferred to as hypertrophic osteoarthritis, osteoarthrosis, anddegenerative joint disease. OA is a chronic degenerative disease ofskeletal joints, which affects specific joints, commonly knees, hips,hand joints and spine, in adults of all ages. OA is characterized by anumber of the following manifestations including degeneration andthinning of the articular cartilage with associated development of“ulcers” or craters, osteophyte formation, hypertrophy of bone at themargins, and changes in the snyovial membrane and enlargement ofaffected joints. Furthermore, osteoarthritis is accompanied by pain andstiffness, particularly after prolonged activity. The antibody, orantigen-binding fragment thereof, of the invention can be used to treatosteoarthritis. Characteristic radiographic features of osteoarthritisinclude joint space narrowing, subchondral sclerosis, osteophytosis,subchondral cyst formation, loose osseous body (or “joint mouse”).

Medications used to treat osteoarthritis include a variety ofnonsteroidal, anti-inflammatory drugs (NSAIDs). In addition, COX2inhibitors, including Celebrex, Vioxx, and Bextra, aand Etoricoxib, arealso used to treat OA. Steroids, which are injected directly into thejoint, may also be used to reduce inflammation and pain. In oneembodiment of the invention, TNFα antibodies of the invention areadministered in combination with a NSAIDs, a COX2 inhibitor, and/orsteroids.

11. Other

The methods of the invention, also can be used to treat various otherdisorders in which TNFα activity is detrimental. Examples of otherdiseases and disorders in which TNFα activity has been implicated in thepathophysiology, and thus which can be treated using an antibody, orantibody portion, of the invention, include inflammatory bone disorders,bone resorption disease, coagulation disturbances, burns, reperfusioninjury, keloid formation, scar tissue formation, pyrexia, periodontaldisease, obesity, radiation toxicity, age-related cachexia, Alzheimer'sdisease, brain edema, inflammatory brain injury, cancer, chronic fatiguesyndrome, dermatomyositis, drug reactions, such as Stevens-Johnsonsyndrome and Jarisch-Herxheimer reaction, edema in and/or around thespinal cord, familial periodic fevers, Felty's syndrome, fibrosis,glomerulonephritides (e.g. post-streptococcal glomerulonephritis or IgAnephropathy), loosening of prostheses, microscopic polyangiitis, mixedconnective tissue disorder, multiple myeloma, cancer and cachexia,multiple organ disorder, myelo dysplastic syndrome, orchitismosteolysis, pancreatitis, including acute, chronic, and pancreaticabscess, polymyositis, progressive renal failure, pseudogout, pyodermagangrenosum, relapsing polychondritis, rheumatic heart disease,sarcoidosis, sclerosing cholangitis, stroke, thoracoabdominal aorticaneurysm repair (TAAA), TNF receptor associated periodic syndrome(TRAPS), symptoms related to Yellow Fever vaccination, inflammatorydiseases associated with the ear, chronic ear inflammation, chronicotitis media with or without cholesteatoma, pediatric ear inflammation,myotosis, ovarian cancer, colorectal cancer, therapy associated withinduced inflammatory syndrome (e.g., syndromes following IL-2administration), and a disorder associated with a reperfussion injury.

It is understood that all of the above-mentioned TNFα-related disordersinclude both the adult and juvenile forms of the disease whereappropriate. It is also understood that all of the above-mentioneddisorders include both chronic and acute forms of the disease. Inaddition, the multiple-variable dose methods of the invention can beused to treat each of the above-mentioned TNFα-related disorders aloneor in combination with one another, e.g., a subject who is sufferingfrom uveitis and lupus.

IV. Pharmaceutical Compositions and Pharmaceutical Administration

A. Compositions and Administration

Antibodies, antibody-portions, and other TNFα inhibitors for use in themultiple-variable dose methods of the invention, can be incorporatedinto pharmaceutical compositions suitable for administration to asubject. Typically, the pharmaceutical composition comprises anantibody, antibody portion, or other TNFα inhibitor of the invention anda pharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it ispreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody, antibody portion, or other TNFα inhibitor.

The compositions for use in the methods of the invention may be in avariety of forms. These include, for example, liquid, semi-solid andsolid dosage forms, such as liquid solutions (e.g., injectable andinfusible solutions), dispersions or suspensions, tablets, pills,powders, liposomes and suppositories. The preferred form depends on theintended mode of administration and therapeutic application. Typicalpreferred compositions are in the form of injectable or infusiblesolutions, such as compositions similar to those used for passiveimmunization of humans with other antibodies or other TNFα inhibitors.The preferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody or other TNFα inhibitor is administered byintravenous infusion or injection. In another preferred embodiment, theantibody or other TNFα inhibitor is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody, antibody portion, or other TNFα inhibitor) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The proper fluidity of a solution can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portionfor use in the methods of the invention is coformulated with and/orcoadministered with one or more additional therapeutic agents. Forexample, an anti-hTNFα antibody or antibody portion of the invention maybe coformulated and/or coadministered with one or more DMARD or one ormore NSAID or one or more additional antibodies that bind other targets(e.g., antibodies that bind other cytokines or that bind cell surfacemolecules), one or more cytokines, soluble TNFα receptor (see e.g., PCTPublication No. WO 94/06476) and/or one or more chemical agents thatinhibit hTNFα production or activity (such as cyclohexane-ylidenederivatives as described in PCT Publication No. WO 93/19751) or anycombination thereof. Furthermore, one or more antibodies of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible side effects, complications or low level of responseby the patient associated with the various monotherapies.

In one embodiment, the invention includes pharmaceutical compositionscomprising an effective amount of a TNFα inhibitor and apharmaceutically acceptable carrier, wherein the effective amount of theTNFα inhibitor may be effective to treat a TNFα-related disorder,including, for example, Crohn's disease, in a multiple variable doseregimen. In one embodiment, the antibody or antibody portion for use inthe multiple variable dose methods of the invention is incorporated intoa pharmaceutical formulation as described in PCT/IB03/04502 and U.S.application Ser. No. 10/222,140, incorporated by reference herein. Thisformulation includes a concentration 50 mg/ml of the antibody D2E7,wherein one pre-filled syringe contains 40 mg of antibody forsubcutaneous injection.

The antibodies, antibody-portions, and other TNFα inhibitors of thepresent invention can be administered by a variety of methods known inthe art, although for many therapeutic applications, the preferredroute/mode of administration is subcutaneous injection. In anotherembodiment, administration is via intravenous injection or infusion. Aswill be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

The TNFα antibodies of the invention can also be administered in theform of protein crystal formulations which include a combination ofprotein crystals encapsulated within a polymeric carrier to form coatedparticles. The coated particles of the protein crystal formulation mayhave a spherical morphology and be microspheres of up to 500 micrometers in diameter or they may have some other morphology and bemicroparticulates. The enhanced concentration of protein crystals allowsthe antibody of the invention to be delivered subcutaneously. In oneembodiment, the TNFα antibodies of the invention are delivered via aprotein delivery system, wherein one or more of a protein crystalformulation or composition, is administered to a subject with aTNFα-related disorder. Compositions and methods of preparing stabilizedformulations of whole antibody crystals or antibody fragment crystalsare also described in WO 02/072636, which is incorporated by referenceherein. In one embodiment, a formulation comprising the crystallizedantibody fragments described in PCT/IB03/04502 and U.S. application Ser.No. 10/222,140, incorporated by reference herein, are used to treat aTNFα-related disorder using the multiple-variable dose methods of theinvention.

In certain embodiments, an antibody, antibody portion, or other TNFαinhibitor of the invention may be orally administered, for example, withan inert diluent or an assimilable edible carrier. The compound (andother ingredients, if desired) may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. To administer a compound ofthe invention by other than parenteral administration, it may benecessary to coat the compound with, or co-administer the compound with,a material to prevent its inactivation.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody portion of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibody,antibody portion, or other TNFα inhibitor may vary according to factorssuch as the disease state, age, sex, and weight of the individual, andthe ability of the antibody, antibody portion, other TNFα inhibitor toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody, antibody portion, or other TNFα inhibitor are outweighed bythe therapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody or antibody portion ofthe invention is 10 to 200 mg, more preferably 20 to 160 mg, morepreferably 40 to 80 mg, and most preferably 80 mg. In one embodiment,the therapeutically effective amount of an antibody or portion thereoffor use in the methods of the invention is about 20 mg. In anotherembodiment, the therapeutically effective amount of an antibody orportion thereof for use in the methods of the invention is about 40 mg.In still another embodiment, the therapeutically effective amount of anantibody or portion thereof for use in the methods of the invention isabout 80 mg. In one embodiment, the therapeutically effective amount ofan antibody or portion thereof for use in the methods of the inventionis about 120 mg. In yet another embodiment, the therapeuticallyeffective amount of an antibody or portion thereof for use in themethods of the invention is about 160 mg. Ranges intermediate to theabove recited dosages, e.g. about 78.5 to about 81.5; about 15 to about25; about 30 to about 50; about 60 to about 100; about 90 to about 150;about 120 to about 200, are also intended to be part of this invention.For example, ranges of values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included.

The invention provides a multiple-variable dose method for treating adisorder in which TNFα activity is detrimental, comprising administeringto a subject in need thereof at least one induction dose of a TNFαinhibitor, such as a human antibody, such that a threshold level of TNFαinhibitor is achieved within an induction phase, and subsequentlyadministering to the subject a treatment dose of the human antibodywithin a treatment phase, such that treatment occurs.

The multiple-variable dose treatment method of the invention comprisesadministering a therapeutic agent in an induction phase, followed by alower amount of the therapeutic agent during a treatment phase. In oneembodiment, the induction dose comprises either 160 mg or 80 mg. Inanother embodiment, the induction dose ranges from about 20 to about 200mg of a TNFα inhibitor. More preferably the induction dose ranges fromabout 40 to about 160 mg of a TNFα inhibitor. Most preferably theinduction dose ranges from about 80 to about 160 mg of a TNFα antibody.The induction phase is complete once a threshold level of therapeuticagent is reached. The induction phase can include a single inductiondose, or multiple induction doses wherein the same or different amountsof therapeutic agent are used. More than one induction dose may beadministered during the induction phase, wherein any determined amountof time interval may occur between induction doses, including, forexample, one hour apart, one day apart, one week apart, two weeks apart,etc. Examples of induction phase treatments of the invention used toachieve a threshold level of TNFα inhibitor include, but are not limitedto, the following regimens: a 160 mg dose followed by an 80 mg dose; atleast one dose of 160 mg dose; at least one dose of 80 mg dose; at leasttwo doses of 80 mg dose; and two 80 mg induction doses at a one weekinterval.

A threshold level is achieved once a pre-determined therapeutic effectis reached. For example, the threshold level of a TNFα inhibitor for thetreatment of Crohn's disease may be determined by monitoring a subjectin the induction phase of treatment for a reduction in their CDAI index.In another example, the threshold level of a TNFα inhibitor fortreatment of psoriasis may be determined by a decrease in psoriaticplaques, an improvement in the patient's Psoriasis Area Severity Index(PAST) score, or an improved Physician's Global Assessment (PGA) score.In still another example, the threshold level of a TNFα inhibitor fortreatment of a TNFα-related disorder is determined by achievement of astable blood plasma serum concentration of the TNFα inhibitor.

Once a threshold level is achieved, the treatment phase is initiated. Atleast one treatment dose is administered during the treatment phase.More than one treatment dose may administered during the treatmentphase, wherein any determined amount of time interval may occur betweeninduction doses, including, for example, one hour apart, one day apart,one week apart, two weeks apart, etc. In one embodiment, the treatmentdose ranges from about 20 to about 120 mg of a TNFα inhibitor. Mostpreferably the treatment dose ranges from about 40 to about 80 mg of aTNFα inhibitor. In one embodiment, the treatment phase comprisesadministering 40 mg of a TNFα inhibitor. In another embodiment, thetreatment phase comprises administering 80 mg of a TNFα inhibitor.

The multiple-variable dose method described herein is based on atreatment regimen which includes administration of at least twodifferent doses of a TNFα inhibitor. The induction dose can be anymultiple number greater than the treatment dose. For example, theinduction dose can be two times greater than the treatment dose. In oneembodiment of the invention, the induction dose is 160 mg, and thetreatment dose is 80 mg. In another embodiment, the induction dose is 80mg, and the treatment dose is 40 mg. In yet another embodiment, theinduction dose is 70 mg, and the treatment dose is 35 mg. In anotherexample, the treatment dose is 40% to 60% of the induction dose, e.g.,the induction dose is 160 mg and the treatment dose ranges from 64 mg to96 mg or the induction dose ranges from 80 mg and the treatment dose is32 mg to 48 mg.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

The invention also pertains to packaged pharmaceutical compositions orkits for administering the multiple-variable dose regimen of theinvention. In one embodiment of the invention, the kit comprises a TNFαinhibitor, such as an antibody, and administration instructionsaccording to the multiple-variable dose method for treatment. In oneembodiment, the kit of the invention comprises an induction dose and/ora treatment dose for treatment of a particular disorder in which TNFαactivity is detrimental. The kit may also include instructions relatingto administration of the induction and/or treatment doses. Theinstructions may describe how, e.g., subcutaneously, and when, e.g., atweek 0 and week 2, the different doses of TNFα inhibitor shall beadministered to a subject for treatment. The instructions may alsodescribe the administration of the TNFα inhibitor during the inductionand the treatment phases of the multiple-variable dose treatment.

Kits to be used for the methods of the invention may include individualdoses of a TNFα inhibitor which can be used in part, in whole, or incombination with one another to achieve the multiple-variable doseregimen. For example, the kit may include a number of prefilled syringescontaining the TNFα antibody D2E7, wherein each syringe contains a 40 mgdose of the TNFα inhibitor. In one embodiment, multiple-variable dosetherapy includes administration of a 160 mg induction dose of D2E7,followed by subsequent administration of an 80 mg dose of D2E7 at leasttwo weeks following administration of the induction dose for thetreatment of Crohn's disease. In such a case, the instructions woulddescribe administration of four syringes of D2E7 for the induction dose,followed by administration of two syringes of D2E7 at least two weekslater. A kit of the invention for the treatment of Crohn's may alsoinclude a dose or doses of methotrexate for administration incombination with D2E7.

In another example, the kit of the invention may include doses of D2E7for multiple-variable dose treatment of psoriasis. In one embodiment,the kit may contain at least one induction dose of 80 mg of D2E7, and atleast one maintenance dose of 40 mg of D2E7. Instructions foradministration of D2E7 for the treatment of psoriasis may include, forexample, directions for administering one 80 mg dose, a second 80 mgdose a week later, and a 40 mg dose a week later and subsequently everyother week. In another example, the instructions may include directionsfor a single 80 mg dose of D2E7, followed by a 40 mg treatment dose aweek later and subsequently every other week.

Another aspect of the invention pertains to kits containing apharmaceutical composition comprising an anti-TNFα antibody and apharmaceutically acceptable carrier and one or more pharmaceuticalcompositions each comprising a drug useful for treating a TNFα-relateddisorder and a pharmaceutically acceptable carrier. Alternatively, thekit comprises a single pharmaceutical composition comprising ananti-TNFα antibody, one or more drugs useful for treating a TNFα-relateddisorder and a pharmaceutically acceptable carrier. The kits containinstructions for dosing of the pharmaceutical compositions for thetreatment of a TNFα-related disorder in which the administration of ananti-TNFα antibody is beneficial, such as Crohn's disease or psoriasis.

The package or kit alternatively can contain the TNFα inhibitor and itcan be promoted for use, either within the package or throughaccompanying information, for the uses or treatment of the disordersdescribed herein. The packaged pharmaceuticals or kits further caninclude a second agent (as described herein) packaged with or copromotedwith instructions for using the second agent with a first agent (asdescribed herein).

In one embodiment, the invention also provides a single dose method fortreating a disorder in which TNFα activity is detrimental, comprisingadministering to a subject in need thereof a single dose of a TNFαinhibitor, such as a human antibody. In one embodiment, the TNFαinhibitor is the anti-TNFα antibody D2E7. The single dose of TNFαinhibitor can be any therapeutically or prophylactically effectiveamount. In one embodiment, a subject is administered either about 20 mg,40 mg, or 80 mg single dose of D2E7. The single dose may be administeredthrough any route, including, for example, subcutaneous administration.

B. Additional Therapeutic Agents

The invention pertains to pharmaceutical compositions and methods of usethereof for the treatment of a TNFα-related disorder using amultiple-variable dose regimen. The pharmaceutical compositions comprisea first agent that prevents or inhibits a TNFα-related disorder. Thepharmaceutical composition and methods of use may comprise a secondagent that is an active pharmaceutical ingredient; that is, the secondagent is therapeutic and its function is beyond that of an inactiveingredient, such as a pharmaceutical carrier, preservative, diluent, orbuffer. The second agent may be useful in treating or preventingTNFα-related disorders. The second agent may diminish or treat at leastone symptom(s) associated with the targeted disease. The first andsecond agents may exert their biological effects by similar or unrelatedmechanisms of action; or either one or both of the first and secondagents may exert their biological effects by a multiplicity ofmechanisms of action. A pharmaceutical composition may also comprise athird compound, or even more yet, wherein the third (and fourth, etc.)compound has the same characteristics of a second agent.

It should be understood that the pharmaceutical compositions describedherein may have the first and second, third, or additional agents in thesame pharmaceutically acceptable carrier or in a differentpharmaceutically acceptable carrier for each described embodiment. Itfurther should be understood that the first, second, third andadditional agent may be administered simultaneously or sequentiallywithin described embodiments. Alternatively, a first and second agentmay be administered simultaneously, and a third or additional agent maybe administered before or after the first two agents.

The combination of agents used within the methods and pharmaceuticalcompositions described herein may have a therapeutic additive orsynergistic effect on the condition(s) or disease(s) targeted fortreatment. The combination of agents used within the methods orpharmaceutical compositions described herein also may reduce adetrimental effect associated with at least one of the agents whenadministered alone or without the other agent(s) of the particularpharmaceutical composition. For example, the toxicity of side effects ofone agent may be attenuated by another agent of the composition, thusallowing a higher dosage, improving patient compliance, and improvingtherapeutic outcome. The additive or synergistic effects, benefits, andadvantages of the compositions apply to classes of therapeutic agents,either structural or functional classes, or to individual compoundsthemselves.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, an antibody or antibody portion ofthe invention is coformulated with and/or coadministered with one ormore additional therapeutic agents that are useful for treatingTNFα-related disorder in which TNFα activity is detrimental. Forexample, an anti-hTNFα antibody, antibody portion, or other TNFαinhibitor of the invention may be coformulated and/or coadministeredwith one or more additional antibodies that bind other targets (e.g.,antibodies that bind other cytokines or that bind cell surfacemolecules), one or more cytokines, soluble TNFα receptor (see e.g., PCTPublication No. WO 94/06476) and/or one or more chemical agents thatinhibit hTNFα production or activity (such as cyclohexane-ylidenederivatives as described in PCT Publication No. WO 93/19751).Furthermore, one or more antibodies or other TNFα inhibitors of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies. Specific therapeutic agent(s) are generallyselected based on the particular TNFα-related disorder being treated, asdiscussed below.

Nonlimiting examples of therapeutic agents with which an antibody,antibody portion, or other TNFα inhibitor can be combined in a multiplevariable dose method of treatment of the invention include thefollowing: non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356(humanized anti-TNFα antibody; Celltech/Bayer); cA2/infliximab (chimericanti-TNFα antibody; Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNFreceptor-IgG fusion protein; Immunex; see e.g., Arthritis & Rheumatism(1994) Vol. 37, S295; J. Invest. Med. (1996) Vol. 44, 235A); 55kdTNF-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche);IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol. 38, S185);DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins; Seragen; seee.g., Arthritis & Rheumatism (1993) Vol. 36, 1223); Anti-Tac (humanizedanti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatorycytokine; DNAX/Schering); IL-10 (SCH 52000; recombinant IL-10,anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4agonists (e.g., agonist antibodies); IL-1RA (IL-1 receptor antagonist;Synergen/Amgen); anakinra (Kineret®/Amgen); TNF-bp/s-TNF (soluble TNFbinding protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), 5284; Amer. J. Physiol.—Heart and Circulatory Physiology(1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase Type IVinhibitor; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S282); MK-966 (COX-2 Inhibitor; see e.g., Arthritis &Rheumatism (1996) Vol. 39, No. 9 (supplement), S81); Iloprost (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S82);methotrexate; thalidomide (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282) and thalidomide-related drugs (e.g.,Celgen); leflunomide (anti-inflammatory and cytokine inhibitor; seee.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S131;Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid(inhibitor of plasminogen activation; see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S284); T-614 (cytokine inhibitor;see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S282); prostaglandin E1 (see e.g., Arthritis & Rheumatism (1996) Vol.39, No. 9 (supplement), S282); Tenidap (non-steroidal anti-inflammatorydrug; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S280); Naproxen (non-steroidal anti-inflammatory drug; seee.g., Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidalanti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatorydrug); Diclofenac (non-steroidal anti-inflammatory drug); Indomethacin(non-steroidal anti-inflammatory drug); Sulfasalazine (see e.g.,Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement), S281);Azathioprine (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S281); ICE inhibitor (inhibitor of the enzymeinterleukin-1(3 converting enzyme); zap-70 and/or lck inhibitor(inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor and/orVEGF-R inhibitor (inhibitos of vascular endothelial cell growth factoror vascular endothelial cell growth factor receptor; inhibitors ofangiogenesis); corticosteroid anti-inflammatory drugs (e.g., SB203580);TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies;interleukin-11 (see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9(supplement), S296); interleukin-13 (see e.g., Arthritis & Rheumatism(1996) Vol. 39, No. 9 (supplement), S308); interleukin-17 inhibitors(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),S120); gold; penicillamine; chloroquine; hydroxychloroquine;chlorambucil; cyclosporine; cyclophosphamide; total lymphoidirradiation; anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;orally-administered peptides and collagen; lobenzarit disodium; CytokineRegulating Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals,Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10;T Cell Sciences, Inc.); prednisone; orgotein; glycosaminoglycanpolysulphate; minocycline; anti-IL2R antibodies; marine and botanicallipids (fish and plant seed fatty acids; see e.g., DeLuca et al. (1995)Rheum. Dis. Clin. North Am. 21:759-777); auranofin; phenylbutazone;meclofenamic acid; flufenamic acid; intravenous immune globulin;zileuton; azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);sirolimus (rapamycin); amiprilose (therafectin); cladribine(2-chlorodeoxyadenosine); methotrexate; antivirals; and immunemodulating agents. Any of the above-mentioned agents can be administeredin combination with the TNFα antibody of the invention to treat anTNFα-related disorder using the multiple variable dose or single dosemethod of treatments of the invention.

In one embodiment, the TNFα antibody of the invention is administered incombination with one of the following agents for the treatment ofrheumatoid arthritis using the multiple variable dose method oftreatment of the invention: small molecule inhibitor of KDR (ABT-123),small molecule inhibitor of Tie-2; methotrexate; prednisone; celecoxib;folic acid; hydroxychloroquine sulfate; rofecoxib; etanercept;infliximab; anakinra (Kineret®/Amgen); leflunomide; naproxen;valdecoxib; sulfasalazine; ibuprofen; methylprednisolone; meloxicam;methylprednisolone acetate; gold sodium thiomalate; aspirin;azathioprine; triamcinolone acetonide; propxyphene napsylate/apap;folate; nabumetone; diclofenac; piroxicam; etodolac; diclofenac sodium;oxaprozin; oxycodone hcl; hydrocodone bitartrate/apap; diclofenacsodium/misoprostol; fentanyl; anakinra, human recombinant; tramadol hcl;salsalate; sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen;alendronate sodium; prednisolone; morphine sulfate; lidocainehydrochloride; indomethacin; glucosamine sulfate/chondroitin;cyclosporine; sulfadiazine; amitriptyline hcl; oxycodonehcl/acetaminophen; olopatadine hcl; misoprostol; naproxen sodium;omeprazole; mycophenolate mofetil; cyclophosphamide; rituximab; IL-1TRAP; MRA; CTLA4-IG; IL-18 BP; ABT-874; ABT-325 (anti-IL 18); anti-IL15; BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;CDC-801; and mesopram. In another embodiment, the TNFα antibody of theinvention is administered using a multiple-variable dose method for thetreatment of a TNFα related disorder in combination with one of theabove mentioned agents for the treatment of rheumatoid arthritis. Inanother embodiment, the above-mentioned additional agents are used incombination with a TNFα antibody in the single dose method of treatmentof the invention.

In one embodiment, the TNFα antibody of the invention is administeredusing the multiple variable dose regimen in combination with one of thefollowing agents for the treatment of a TNFα-related disorder in whichTNFα activity is detrimental: anti-IL12 antibody (ABT 874); anti-IL18antibody (ABT 325); small molecule inhibitor of LCK; small moleculeinhibitor of COT; anti-IL1 antibody; small molecule inhibitor of MK2;anti-CD19 antibody; small molecule inhibitor of CXCR3; small moleculeinhibitor of CCR5; small molecule inhibitor of CCR11 anti-E/L selectinantibody; small molecule inhibitor of P2X7; small molecule inhibitor ofIRAK-4; small molecule agonist of glucocorticoid receptor; anti-05areceptor antibody; small molecule inhibitor of C5a receptor; anti-CD32antibody; and CD32 as a therapeutic protein.

In yet another embodiment, the TNFα antibody of the invention isadministered using the multiple variable dose regimen in combinationwith an antibiotic or antiinfective agent. Antiinfective agents includethose agents known in the art to treat viral, fungal, parasitic orbacterial infections. The term, “antibiotic,” as used herein, refers toa chemical substance that inhibits the growth of, or kills,microorganisms. Encompassed by this term are antibiotic produced by amicroorganism, as well as synthetic antibiotics (e.g., analogs) known inthe art. Antibiotics include, but are not limited to, clarithromycin(Biaxin®), ciprofloxacin (Cipro®), and metronidazole (Flagyl®).

In another embodiment, the TNFα antibody of the invention isadministered using the multiple variable dose regimen in combinationwith an additional therapeutic agent to treat sciatica or pain. Examplesof agents which can be used to reduce or inhibit the symptoms ofsciatica or pain include hydrocodone bitartrate/apap, rofecoxib,cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen, oxycodonehcl/acetaminophen, celecoxib, valdecoxib, methylprednisolone acetate,prednisone, codeine phosphate/apap, tramadol hcl/acetaminophen,metaxalone, meloxicam, methocarbamol, lidocaine hydrochloride,diclofenac sodium, gabapentin, dexamethasone, carisoprodol, ketorolactromethamine, indomethacin, acetaminophen, diazepam, nabumetone,oxycodone hcl, tizanidine hcl, diclofenac sodium/misoprostol,propoxyphene napsylate/apap, asa/oxycod/oxycodone ter,ibuprofen/hydrocodone bit, tramadol hcl, etodolac, propoxyphene hcl,amitriptyline hcl, carisoprodol/codeine phos/asa, morphine sulfate,multivitamins, naproxen sodium, orphenadrine citrate, and temazepam.

In yet another embodiment, the TNFα-related disorder is treated usingthe multiple variable dose regimen with the TNFα antibody of theinvention in combination with hemodialysis.

In another embodiment, a TNFα antibody of the invention is used incombination with a drug used to treat Crohn's disease or aCrohn's-related disorder in the multiple variable dose regimen of theinvention. Examples of therapeutic agents which can be used to treatCrohn's include mesalamine, prednisone, azathioprine, mercaptopurine,infliximab, budesonide, sulfasalazine, methylprednisolone sod succ,diphenoxylate/atrop sulf, loperamide hydrochloride, methotrexate,omeprazole, folate, ciprofloxacin/dextrose-water, hydrocodonebitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, hyoscyamine sulfate,cholestyramine/sucrose, ciprofloxacin hydrochloride, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, natalizumab, methylprednisolone, interferon-gamma, andsargramostim (GM-CSF). In one embodiment, methotrexate is administeredfor the treatment of Crohn's disease at a dose of 2.5 mg to 30 mg perweek.

In another embodiment, a TNFα antibody is administered in combinationwith an additional therapeutic agent to treat asthma in the multiplevariable dose regimen of the invention. Examples of agents which can beused to reduce or inhibit the symptoms of asthma include the following:albuterol; salmeterol/fluticasone; sodium; fluticasone propionate;budesonide; prednisone; salmeterol xinafoate; levalbuterol hcl;sulfate/ipratropium; prednisolone sodium phosphate; triamcinoloneacetonide; beclomethasone dipropionate; ipratropium bromide;Azithromycin; pirbuterol acetate; prednisolone; theophylline anhydrous;zafirlukast; methylprednisolone sod succ; clarithromycin; formoterolfumarate; influenza virus vaccine; methylprednisolone; trihydrate;allergy injection; cromolyn sodium; cefprozil; fexofenadinehydrochloride; flunisolide/menthol; levofloxacin;amoxicillin/clavulanate, inhaler assist device, guaifenesin,dexamethasone sod phosphate; moxifloxacin hcl; hyclate;guaifenesin/d-methorphan; gatifloxacin; pephedrine/cod/chlorphenir;cetirizine hydrochloride; mometasone furoate; salmeterol xinafoate;benzonatate; cephalexin; pe/hydrocodone/chlorphenir; cetirizinehcl/pseudoephed; phenylephrine/cod/promethazine; codeine/promethazine;flunisolide; dexamethasone; guaifenesin/pseudoephedrine;chlorpheniramine/hydrocodone; nedocromil sodium; terbutaline sulfate;epinephrine and methylprednisolone, metaproterenol sulfate.

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat COPD in the multiple variable dose regimen of the invention.Examples of agents which can be used to reduce or inhibit the symptomsof COPD include, albuterol sulfate/ipratropium; ipratropium bromide;salmeterol/fluticasone; albuterol; salmeterol; xinafoate; fluticasonepropionate; prednisone; theophylline anhydrous; levofloxacin;methylprednisolone sod succ; montelukast sodium; budesonide; formoterolfumarate; triamcinolone acetonide; guaifenesin; azithromycin;beclomethasone; dipropionate; levalbuterol hcl; flunisolide; sodium;trihydrate; gatifloxacin; zafirlukast; furoate; amoxicillin/clavulanate;flunisolide/menthol; chlorpheniramine/hydrocodone; metaproterenolsulfate; methylprednisolone; ephedrine/cod/chlorphenir; pirbuterolacetate; -ephedrine/loratadine; terbutaline sulfate; tiotropium bromide;(R,R)-formoterol; TgAAT; Cilomilast and Roflumilast

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat IPF in the multiple variable dose regimen of the invention.Examples of agents which can be used to reduce or inhibit the symptomsof IPF include prednisone; azathioprine; albuterol; colchicines;sulfate; digoxin; gamma interferon; methylprednisolone sod succ;furosemide; lisinopril; nitroglycerin; spironolactone; cyclophosphamide;ipratropium bromide; actinomycin d; alteplase; fluticasone propionate;levofloxacin; metaproterenol sulfate; morphine sulfate; oxycodone hcl;potassium chloride; triamcinolone acetonide; tacrolimus anhydrous;calcium; interferon-alpha; methotrexate; mycophenolate mofetil.

In one embodiment of the invention, a TNFα antibody is administered incombination with an agent which is commonly used to treatspondyloarthropathies in the multiple variable dose regimen of theinvention. Examples of such agents include nonsteroidal,anti-inflammatory drugs (NSAIDs), COX 2 inhibitors, including Celebrex®,Vioxx®, and Bextra®, aand etoricoxib. Physiotherapy is also commonlyused to treat spondyloarthropathies, usually in conjunction withnon-steoidal inflammatory drugs.

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat ankylosing spondylitis in the multiple variable dose regimen ofthe invention. Examples of agents which can be used to reduce or inhibitthe symptoms of ankylosing spondylitis include ibuprofen, diclofenac andmisoprostol, naproxen, meloxicam, indomethacin, diclofenac, celecoxib,rofecoxib, sulfasalazine, prednisone, methotrexate, azathioprine,minocyclin, prednisone, etanercept, and infliximab.

In another embodiment, the TNFα antibody of the invention isadministered in combination with an additional therapeutic agent totreat psoriatic arthritis in the multiple variable dose regimen of theinvention. Examples of agents which can be used to reduce or inhibit thesymptoms of psoriatic arthritis include methotrexate; etanercept;rofecoxib; celecoxib; folic acid; sulfasalazine; naproxen; leflunomide;methylprednisolone acetate; indomethacin; hydroxychloroquine sulfate;sulindac; prednisone; betamethasone diprop augmented; infliximab;methotrexate; folate; triamcinolone acetonide; diclofenac;dimethylsulfoxide; piroxicam; diclofenac sodium; ketoprofen; meloxicam;prednisone; methylprednisolone; nabumetone; tolmetin sodium;calcipotriene; cyclosporine; diclofenac; sodium/misoprostol;fluocinonide; glucosamine sulfate; gold sodium thiomalate; hydrocodone;bitartrate/apap; ibuprofen; risedronate sodium; sulfadiazine;thioguanine; valdecoxib; alefacept; and efalizumab.

In one embodiment the TNFα inhibitor is administered following aninitial procedure for treating coronary heart disease in the multiplevariable dose regimen of the invention. Examples of such proceduresinclude, but are not limited to coronary artery bypass grafting (CABG)and Percutaneous transluminal coronary balloon angioplasty (PTCA) orangioplasty. In one embodiment, the TNFα inhibitor is administered inorder to prevent stenosis from re-occurring. In another embodiment ofthe invention, the TNFα inhibitor is administered in order to prevent ortreat restenosis. The invention also provides a method of treatment,wherein the TNFα inhibitor is administered prior to, in conjunctionwith, or following the insertion of a stent in the artery of a subjectreceiving a procedure for treating coronary heart disease. In oneembodiment the stent is administered following CABG or PTCA.

A wide variety of stent grafts may be utilized within the context of thepresent invention, depending on the site and nature of treatmentdesired. Stent grafts may be, for example, bifurcated or tube grafts,cylindrical or tapered, self-expandable or balloon-expandable, unibody,or, modular. Moreover, the stent graft may be adapted to release thedrug at only the distal ends, or along the entire body of the stentgraft. The TNFα inhibitor of the invention can also be administered on astent. In one embodiment, the TNFα antibody of the invention, including,for example, D2E7/HUMIRA® is administered by a drug-eluting stent.

The TNFα antibody can be administered in combination with an additionaltherapeutic agent to treat restenosis in the multiple variable doseregimen of the invention. Examples of agents which can be used to treator prevent restenosis include sirolimus, paclitaxel, everolimus,tacrolimus, ABT-578, and acetaminophen.

The TNFα antibody of the invention can be administered in combinationwith an additional therapeutic agent to treat myocardial infarction inthe multiple variable dose regimen of the invention. Examples of agentswhich can be used to treat or prevent myocardial infarction includeaspirin, nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparinsodium, clopidogrel bisulfate, carvedilol, atenolol, morphine sulfate,metoprolol succinate, warfarin sodium, lisinopril, isosorbidemononitrate, digoxin, furosemide, simvastatin, ramipril, tenecteplase,enalapril maleate, torsemide, retavase, losartan potassium, quinaprilhcl/mag carb, bumetanide, alteplase, enalaprilat, amiodaronehydrochloride, tirofiban hcl m-hydrate, diltiazem hydrochloride,captopril, irbesartan, valsartan, propranolol hydrochloride, fosinoprilsodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium,atropine sulfate, aminocaproic acid, spironolactone, interferon, sotalolhydrochloride, potassium chloride, docusate sodium, dobutamine hcl,alprazolam, pravastatin sodium, atorvastatin calcium, midazolamhydrochloride, meperidine hydrochloride, isosorbide dinitrate,epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,ezetimibe/simvastatin, avasimibe, abciximab, and cariporide.

The TNFα antibody of the invention can be administered in combinationwith an additional therapeutic agent to treat angina in the multiplevariable dose regimen of the invention. Examples of agents which can beused to treat or prevent angina include: aspirin; nitroglycerin;isosorbide mononitrate; atenolol; metoprolol succinate; metoprololtartrate; amlodipine besylate; digoxin; dilitiazem hydropchloride;isosorbide dinitrate; clopidogrel bisulfate; nifedipine; atorvastatincalcium; potassium chloride; simvastatin; verapamil hcl; furosemide;propranolol hcl; carvedilo; lisinopril; sprionolactone;hydrochlorothiazide; enalapril maleate; madolol; ramipril; enoxaparinsodium; heparin sodium; valsartan; sotalol hydrochloride; fenofibrate;ezetimibe; bumetanide; losartan potassium;lisinopril/hydrochlorothiazide; felodipine; captopril; and bisoprololfumarate.

In one embodiment of the invention, a TNFα antibody is administered incombination with an agent which is commonly used to treat hepatitis Cvirus in the multiple variable dose regimen of the invention. Examplesof such agents include Interferon-aplha-2a, Interferon-alpha-2b,Interferon-alpha con1, Interfero-aopha-n1, Pegylatedinterferon-alpha-2a, Pegylated interferon-alpha-2b, Ribavirin,Peginterferon alfa-2b and ribavirin, Ursodeoxycholic Acid, GlycyrrhizicAcid, Thymalfasin, Maxamine, and VX-497.

The TNFα antibody of the invention is administered in combination withtopical corticosteroids, vitamin D analogs, and topical or oralretinoids, or combinations thereof, for the treatment of psoriasis inthe multiple variable dose regimen of the invention. In addition, theTNFα antibody of the invention is administered in combination with oneof the following agents for the treatment of psoriasis: small moleculeinhibitor of KDR (ABT-123), small molecule inhibitor of Tie-2,calcipotriene, clobetasol propionate, triamcinolone acetonide,halobetasol propionate, tazarotene, methotrexate, fluocinonide,betamethasone diprop augmented, fluocinolone, acetonide, acitretin, tarshampoo, betamethasone valerate, mometasone furoate, ketoconazole,pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide, urea,betamethasone, clobetasol propionate/emoll, fluticasone propionate,azithromycin, hydrocortisone, moisturizing formula, folic acid,desonide, coal tar, diflorasone diacetate, etanercept, folate, lacticacid, methoxsalen, hc/bismuth subgal/znox/resor, methylprednisoloneacetate, prednisone, sunscreen, salicylic acid, halcinonide, anthralin,clocortolone pivalate, coal extract, coal tar/salicylic acid, coaltar/salicylic acid/sulfur, desoximetasone, diazepam, emollient,pimecrolimus emollient, fluocinonide/emollient, mineral oil/castoroil/na lact, mineral oil/peanut oil, petroleum/isopropyl myristate,psoralen, salicylic acid, soap/tribromsalan, thimerosal/boric acid,celecoxib, infliximab, alefacept, efalizumab, tacrolimus, pimecrolimus,PUVA, UVB and other phototherapy, and sulfasalazine.

An antibody, antibody portion, or other TNFα inhibitor of the inventioncan be used in combination with other agents to treat skin conditions inthe multiple variable dose regimen of the invention. For example, anantibody, antibody portion, or other TNFα inhibitor of the invention iscombined with PUVA therapy. PUVA is a combination of psoralen (P) andlong-wave ultraviolet radiation (UVA) that is used to treat manydifferent skin conditions. The antibodies, antibody portions, or otherTNFα inhibitors of the invention can also be combined with pimecrolimus.In another embodiment, the antibodies of the invention are used to treatpsoriasis, wherein the antibodies are administered in combination withtacrolimus. In a further embodiment, tacrolimus and TNFα inhibitors areadministered in combination with methotrexate and/or cyclosporine. Instill another embodiment, the TNFα inhibitor of the invention isadministered with excimer laser treatment for treating psoriasis.

Nonlimiting examples of other therapeutic agents with which a TNFαinhibitor can be combined to treat a skin or nail disorder include UVAand UVB phototherapy in the multiple variable dose regimen of theinvention. Other nonlimiting examples which can be used in combinationwith a TNFα inhibitor include anti-IL-12 and anti-IL-18 therapeuticagents, including antibodies.

In one embodiment, the TNFα antibody of the invention is administered incombination with an additional therapeutic agent in the treatment ofBehcet's disease in the multiple variable dose regimen of the invention.Additional therapeutic agents which can be used to treat Behcet'sdisease include, but are not limited to, prednisone, cyclophosphamide(Cytoxan), Azathioprine (also called imuran, methotrexate,timethoprim/sulfamethoxazole (also called bactrim or septra) and folicacid.

Any one of the above-mentioned therapeutic agents, alone or incombination therewith, can be administered to a subject suffering from aTNFα-related disorder in which TNFα is detrimental, in combination withthe TNFα antibody using a multiple variable dose treatment regimen ofthe invention. In one embodiment, any one of the above-mentionedtherapeutic agents, alone or in combination therewith, can beadministered to a subject suffering from rheumatoid arthritis inaddition to a TNFα antibody to treat a TNFα-related disorder. It shouldbe understood that the additional therapeutic agents can be used incombination therapy as described above, but also may be used in otherindications described herein wherein a beneficial effect is desired.

It also is understood that the above-mentioned additional agents canalso be used in combination with a TNFα inhibitor, e.g., a TNFαantibody, to treat a TNFα-related disorder using the single dosetreatment method of the invention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference

EXAMPLES Example 1 Study of Efficacy of Multiple-Dose Therapy forTreatment of Crohn's Disease

Multiple-Variable Dose Treatment of Crohn's Disease With D2E7

Studies were performed to determine the efficacy of a multiple-variabledose regimen of a TNFα inhibitor, namely D2E7 (also referred to asadalimumab and Humir®), for treating Crohn's disease. Efficacy andtolerability of D2E7 in the treatment of patients with active Crohn'sdisease were evaluated in a randomized, double-blind,placebo-controlled, multicenter study.

In this study, two hundred ninety-nine patients without previousexposure to TNF-antagonists and with active Crohn's Disease wereselected. Crohn's disease in each patient was confirmed by endoscopic orradiologic evaluation. Subjects were randomized equally to one of fourtreatment groups (three treatment groups and one placebo group).Eligible subjects included men and women between 18 and 75 years of agehaving a diagnosis of Crohn's disease for more than four months. Inaddition, selected patients had active Crohn's disease, defined as aCrohn's Disease Activity Index (CDAI) score of 220 to 450 points.

At baseline (Week 0), subjects received a loading dose of D2E7 followedby a treatment dose at Week 2, wherein the treatment dose was lower thanthe initial loading dose. Patients received one of the followingmultiple variable dose treatment regimens at Week 0 (baseline) and Week2 (Week 0/Week 2): 160 mg/80 mg D2E7; 80 mg/40 mg D2E7; 40 mg/20 mgD2E7; or placebo/placebo. Patients were administered D2E7 or placebotreatment subcutaneously

The study was conducted for up to ten weeks, including an initial twoweek screening period, a four week treatment period (Weeks 0 to 4), anda four week follow-up period. Participants were evaluated for inductionof clinical remission of Crohn's disease, defined as a CDAI score of<150 at week 4. Clinical response, defined as a decrease in CDAIcompared to the CDAI baseline reading of ≧70 [A70] or ≧100 points [Δ100]), was also assessed in the participants. Efficacy of themultiple-variable dose regimen was further measured according toimprovements in the patient's Inflammatory Bowel Disease Questionnaire(IBDQ) score, and improvements or remission of draining fistulas.Fistula remission was defined as closure of all fistulas that weredraining at baseline for at least two consecutive visits. Fistulaimprovement was defined as a decrease of ≧50% in the number of drainingfistula for at least two consecutive visits. C-reactive protein (CRP)levels were also measured, as CRP levels are reflective of inflammationin the body.

Results from the study show that multiple, variable D2E7 dosagetreatments were effective at inducing Crohn's disease remission. Table 1shows the percentage of patients with clinical remission (CDAI<150) atweek 4 of the dosing regimen. As shown below in Table 1, thirty percentof patients who received 80/40 mg or 160/80 mg of D2E7 achieved clinicalremission compared with 12% who received placebo (p=0.004.) Patients inthe highest dose group, 160/80 mg, achieved a statistically significantremission rate of 36% versus a placebo rate of 12%.

TABLE 1 D2E7 induces clinical remission in treatment groups at Week 4Placebo 40/20 mg 80/40 mg 160/80 mg CDAI ≦ 150 12% 18% 24% 36%*(*denotes p = 0.001) (Placebo n = 74; 20 mg n = 74; 40 mg n = 75; 80 mgn = 76)

Results of remission of Crohn's disease (measured CDAI<150) from eachdosage group are also shown in FIG. 1 (note dosage references in FIG. 1,as well as FIGS. 2-6 refer to the treatment dose, i.e., 40 mg refers tothe 80/40 treatment regimen).

The median 4-week changes in the CDAI index for each dose group (pointswith data at both baseline and week 4) were as follows: placebo, Δ− 47(n=67); 20 mg, Δ−73 (n=70); 40 mg, Δ− 90 (n=70); and 80 mg, Δ− 101(n=73). The decrease in the CDAI index for patients who received themultiple variable dose treatment of D2E7 is also shown in FIG. 2.Clinical response results of CDAI≧70 point and ≧100 point decrease frombaseline at four weeks are shown in FIGS. 3 and 4, respectively. Thus,patients who received multiple, variable doses of D2E7, especiallyCrohn's patients receiving 80/40 mg and 160/80 mg, showed a decrease inthe CDAI index indicating remission of Crohn's disease. In addition, asshown in FIG. 5, patients receiving the 160/80 mg dosing regimen showedthe greatest decrease in CRP levels, with the placebo group showing theleast decrease. Patients also showed an overall improvement in theirIBDQ score, as shown in FIG. 6. In addition, sustained serum D2E7concentrations were achieved early in treatment as a result of themultiple variable dose regimen.

The overall incidence of adverse events (AE) was low and did not differamong groups. The most common AE were injection site reactions, most ofwhich were mild. Statistically significant results were not dependent onbaseline CRP concentration.

In sum, multiple, variable doses of D2E7 significantly increased thefrequency of remission of disease in Crohn's disease subjects. Incombination, 30% of subjects receiving D2E7 doses of 80/40 mg and 160/80mg achieved remission in comparison to only 12% of placebo subjects.There was also a significant increase in the clinical response (adecrease in the CDAI index of ≧70 points) and IBDQ scores of the 40 mgand 80 mg treatment doses every other week compared to the placebo. Inthe treatment group receiving an 80 mg D2E7 treatment dose every otherweek, 49% of the subjects achieved a clinical response (a decrease inthe CDAI of ≧100 points).

Example 2 Additional Study of Efficacy of Multiple-Dose Therapy forTreatment of Crohn's Disease

Multiple-Variable Dose Treatment of Crohn's Disease with D2E7

A study was performed to assess the tolerability and clinical benefit ofa multiple-variable dose treatment using a TNFα inhibitor, specificallyD2E7, in adult patients with Crohn's disease who had previously receivedand responded to a different TNFα inhibitor. The study included patientswho had previously received the chimeric anti-TNF antibody infliximab,but who no longer have a sustained response and/or tolerance toinfliximab.

Patients who had lost responsiveness or developed intolerance (acute ordelayed infusion reactions) were treated with D2E7 80 mg at week 0 and40 mg at week 2. All treatments were subcutaneous. Antibodies toinfliximab (ATI) were determined at baseline (Prometheus Laboratories,San Diego, Calif.). Crohn's disease activity index (CDAI) scores,presence of fistulas, and C-reactive protein (CRP) concentration weredetermined at weeks 0 and 4. Clinical response (decrease in CDAIof >/=100 points), clinical remission (CDAI</=150 points), fistulaimprovement (closure>/=50% of open fistulas), complete fistula closure,and acute and delayed hypersensitivity reactions were recordedthroughout the study.

Twenty-four patients were enrolled and completed 4 weeks of themultiple-variable dose therapy. Four of 16 patients (25%) were positivefor ATI. Of 13 patients with week 0 CDAI scores>/=220, 6 (46%) achievedclinical response and 1 (8%) achieved remission at week 4. Of 6 patientswith perianal and/or rectovaginal fistulas, 4 (67%) had fistulaimprovement and 3 (50%) had complete fistula closure at week 4. Only 6patients (38%) had CRP values above the normal range. Among allpatients, the mean+/−SD CRP concentrations decreased from 17.0+/−29.3mg/L at week 0 to 11.3+/−17.3 mg/L at week 4. No patients experiencedacute or delayed hypersensitivity reactions during treatment with D2E7(including 8 who previously experienced treatment-limiting acutehypersensitivity reactions and 3 who previously experienced delayedhypersensitivity reactions with infliximab).

In sum, multiple-variable dose treatment using D2E7 was well toleratedand was clinically beneficial in patients with Crohn's disease who hadpreviously received and responded to infliximab, but who no longer had asustained response to or could not tolerate infliximab.

Example 3 Efficacy of Multiple-Dose Therapy Using TNFα Inhibitor forTreatment of Psoriasis

Multiple-Variable Dose Treatment of Psoriasis with D2E7

A study was performed to determine the efficacy of a multiple-variabledose regimen of D2E7 for treating psoriasis. Efficacy and tolerabilityof D2E7 in the treatment of patients with moderate to severe chronicplaque psoriasis were evaluated in a randomized, double-blind,placebo-controlled multicenter study.

In this study, one hundred forty-eight adult patients with a diagnosisof moderate to severe psoriasis for at least one year were selected toreceive multiple-variable dose treatment. Patients were also selectedbased on an affected body surface area (BSA) of ≧5%. Subjects wererandomized equally to one of three groups (two treatment groups and oneplacebo).

At baseline (Week 0) patients in both treatment groups received aninduction dose of 80 mg of D2E7. Patients in the first treatment groupsubsequently received a treatment dose of 40 mg of D2E7 at week 1followed by 40 mg every other week (eow) starting at week 3. Subjects inthe second treatment group received an induction dose of 80 mg dose ofD2E7 at Week 1 (following the initial 80 mg dose at Week 0) followed bya treatment dose of 40 mg of D2E7 weekly starting at week 2. The placebogroup received only the placebo weekly starting at baseline. Alltreatment was administered subcutaneously (sc) with pre-filled syringes.A summary of the different regimens are described below in Table 2:

TABLE 2 Psoriasis study regimens Regimen Detailed description A D2E7 80mg sc administered at Week 0 (baseline); D2E7 40 mg sc every other weekadministered starting at Week 1 through Week 11, with placeboadministered on alternate weeks B D2E7 80 mg sc administered starting atWeek 0 (baseline) and at Week 1 (80/80); D2E7 40 mg sc weeklyadministered starting at Week 2 through Week 11 C Placebo sc will beadministered at baseline and then weekly through Week 11, with twoinjections given at Week 1 and Week 1

In order to maintain the blind study, all subjects received a total of 2injections at baseline and week 1. During the remaining period of thestudy (weeks 2 through 12), subjects received one injection per week.The treatment dose per injection correlated to the dose regimen randomlyassigned to each subject.

The PASI of the participants of the multiple-variable dose regimen wasdetermined according to standard methods (see Fredriksson andPettersson, supra and Marls et al., supra). The primary efficacyendpoint of the study was the percentage of subjects achieving aclinical response as defined by at least a 75% reduction in the PASIscore (≧PAST 75) at Week 12.

Secondary efficacy measures included a static Physician's GlobalAssessment (PGA) of “clear” or “almost clear” at Week 12. PGA wasdetermined according to a seven point scale used to measure the severityof psoriasis at the time of the physician's evaluation. Descriptions ofthe disease used included the following: severe=very marked plaqueelevation, scaling, and/or erythema; moderate to severe=marked plaqueelevation, scaling, and/or erythema; moderate=moderate plaque elevation,scaling, and/or erythema; mild to moderate=intermediate between moderateand mild; mild=slight plaque elevation, scaling, and/or erythema; almostclear=intermediate between mild and clear; and clear=no signs ofpsoriasis.

The results show that at Week 12, statistically significantly greaterpercentages of patients achieved a PASI 75 response or better on D2E7than those on a placebo treatment. For patients receiving 40 mgtreatment dose of D2E7 eow, 53% demonstrated a PASI of 75 or higher. Inaddition, 80% of patients receiving a 40 mg treatment dose of D2E7weekly showed a PASI 75 or higher, compared to only 4% of the placebotreatment group (p<0.001 vs. placebo). Response rates at Week 12 forboth dosing regimens of D2E7 were statistically significantly greaterthan for placebo, as shown in FIG. 7.

Overall, the mean percentage changes in PASI score for patients on D2E7multiple-variable dose therapy were statistically significantly greaterthan placebo. The changes were evident as early as Week 1 after theinitial dose, as shown in FIG. 8. At Week 12, 49% of patients on D2E7receiving 40 mg eow and 76% of patients receiving 40 mg of D2E7 weeklyachieved a PGA of “clear” or “almost clear,” compared with 2% of placebopatients.

Of the one hundred forty-eight adult patients enrolled in the study, 29%also had a medical history of psoriatic arthritis (PsA). Both doses ofD2E7 were effective in the treatment of psoriasis in both patients withand without PsA. Patients with PsA had a similar efficacy response toD2E7 as those without PsA. For both PsA and without PsA subgroups, thepercentages of patients achieving a PASI 75 response or better at week12 was statistically significant for the eow (with PsA, 47%; withoutPsA, 57%) and weekly treatment arms (with PsA, 58%; without PsA, 87%)compared with placebo. Continued improvements in efficacy were seenthrough week 24 in the eow arm (with PsA, 53%; without PsA, 70%).Efficacy responses in patients with and without PsA at weeks 12 and 24are shown in FIGS. 9 and 10, respectively.

In conclusion, D2E7 administered for 12 weeks was effective in thetreatment of moderate to severe chronic plaque psoriasis. 53% ofpatients on 40 mg eow achieved ≧PASI 75, compared with 4% on placebo.80% of patients on 40 mg weekly achieved ≧PASI 75. 49% and 76% ofpatients on D2E7 40 mg eow and 40 mg weekly, respectively, were “clear”or “almost clear” of their psoriasis. In addition, D2E7 was equallyeffective at treating psoriasis patients with and without PsA.

Example 4 Efficacy of Single Dose Treatment of D2E7

A study was performed to determine the efficacy of a single dose regimenof D2E7 for treating rheumatoid arthritis (RA). The objective of thestudy was to determine and compare the single-dose safety and efficacyof 3 subcutaneous (sc) doses (20, 40, or 80 mg) of D2E7 in Japanese andCaucasian subjects with RA.

D2E7 was administered as single sc doses (20, 40, or 80 mg) in 40Japanese (in Japan) and 36 Caucasian (in US) subjects with RA,well-matched for moderate-to-severe baseline disease severity, in 2separate clinical studies of similar design—open-label, parallel group.On Study Days 1, 15 and 29, safety evaluations included physicalexaminations, vital signs, and laboratory assessments to determineadverse events (AEs), and efficacy evaluations included CRP, Physician'sand Subject's Assessment of Disease Activity, Subject's Assessment ofPain, Disability Index of the Health Assessment Questionnaire (DIHAQ),and tender and swollen joint counts.

Results from the study showed that all Japanese treatment groups hadstatistically significant improvements of all ACR components (exceptDIHAQ) on Day 15 and on Day 29 compared to Day 1. In the 3 Caucasiantreatment groups, only the 80-mg treatment group exhibited astatistically significant improvement at Day 29 in all individual ACRcomponents with the exception of the DIHAQ score. Although the studyduration was only 29 days, ACR20 responses were achieved in 47.5%(19/40) of the Japanese patients and in 30.6% (11/36) of the Caucasianpatients. In addition, the difference in frequency of subjects reportingAEs between treatment groups was not clinically relevant within eachstudy. Interestingly, there was an increased incidence of AEs inJapanese subjects which may reflect racial differences or investigatorcultural tendency to report.

The results demonstrate an improvement of comparable magnitude in RAsigns and symptoms in both groups in this short-term study using asingle dose treatment. These results also suggest similar safety ofsingle-dose sc administration of ADA in Japanese and Caucasian subjects.

Forming part of the present disclosure is the appended Sequence Listing,the contents of which are summarized in the table below:

ANTIBODY SEQ ID NO: CHAIN REGION SEQUENCE TYPE 1 D2E7 VL amino acid 2D2E7 VH amino acid 3 D2E7 VL CDR3 amino acid 4 D2E7 VH CDR3 amino acid 5D2E7 VL CDR2 amino acid 6 D2E7 VH CDR2 amino acid 7 D2E7 VL CDR1 aminoacid 8 D2E7 VH CDR1 amino acid 9 2SD4 VL amino acid 10 2SD4 VH aminoacid 11 2SD4 VL CDR3 amino acid 12 EP B12 VL CDR3 amino acid 13 VL10E4VL CDR3 amino acid 14 VL100A9 VL CDR3 amino acid 15 VLL100D2 VL CDR3amino acid 16 VLL0F4 VL CDR3 amino acid 17 LOE5 VL CDR3 amino acid 18VLLOG7 VL CDR3 amino acid 19 VLLOG9 VL CDR3 amino acid 20 VLLOH1 VL CDR3amino acid 21 VLLOH10 VL CDR3 amino acid 22 VL1B7 VL CDR3 amino acid 23VL1C1 VL CDR3 amino acid 24 VL0.1F4 VL CDR3 amino acid 25 VL0.1H8 VLCDR3 amino acid 26 LOE7.A VL CDR3 amino acid 27 2SD4 VH CDR3 amino acid28 VH1B11 VH CDR3 amino acid 29 VH1D8 VH CDR3 amino acid 30 VH1A11 VHCDR3 amino acid 31 VH1B12 VH CDR3 amino acid 32 VH1E4 VH CDR3 amino acid33 VH1F6 VH CDR3 amino acid 34 3C-H2 VH CDR3 amino acid 35 VH1-D2.N VHCDR3 amino acid 36 D2E7 VL nucleic acid 37 D2E7 VH nucleic acid

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A multiple-variable dose method for treatingidiopathic inflammatory bowel disease in a human subject in needthereof, comprising subcutaneously administering to the human subject: afirst dose of 160 mg of adalimumab administered to the human subjectwithin a day; and a second dose of 80 mg of adalimumab administered tothe human subject within a day, wherein the second dose is administeredtwo weeks following administration of the first dose.
 2. The method ofclaim 1, wherein the method further comprises administering to the humansubject a subsequent subcutaneous injection of 40 mg of adalimumab twoweeks following administration of the second dose.
 3. The method ofclaim 2, wherein the method further comprises administering to the humansubject additional subsequent subcutaneous injections of 40 mg ofadalimumab, wherein the subsequent subcutaneous injections areadministered two weeks apart.
 4. A multiple-variable dose method fortreating idiopathic inflammatory bowel disease in a human subject inneed thereof, comprising subcutaneously administering to the humansubject: a first dose of 160 mg of adalimumab administered as a set offour injections of 40 mg of adalimumab administered to the human subjectwithin a day; and a second dose of 80 mg of adalimumab administered as aset of two injections of 40 mg of adalimumab administered to the humansubject within a day, wherein the second dose is administered two weeksfollowing administration of the first dose.
 5. The method of claim 4,wherein the method further comprises administering to the human subjecta subsequent subcutaneous injection of 40 mg of adalimumab two weeksfollowing administration of the second dose.
 6. The method of claim 5,wherein the method further comprises administering to the human subjectadditional subsequent subcutaneous injections of 40 mg of adalimumab,wherein the subsequent subcutaneous injections are administered twoweeks apart.
 7. The method of claim 4, wherein each subcutaneousinjection is administered to the human subject using a prefilledsyringe.
 8. The method of claim 5, wherein each subcutaneous injectionis administered to the human subject using a prefilled syringe.
 9. Themethod of claim 1, wherein the human subject has Crohn's disease. 10.The method of claim 1, wherein the human subject has ulcerative colitis.11. The method of claim 4, wherein the human subject has Crohn'sdisease.
 12. The method of claim 4, wherein the human subject hasulcerative colitis.
 13. The method of claim 9, wherein the human subjectachieves a Crohn's Disease Activity Index (CDAI) score of <150.
 14. Themethod of claim 11, wherein the human subject achieves a Crohn's DiseaseActivity Index (CDAI) score of <150.
 15. The method of claim 2, whereinthe human subject has Crohn's disease.
 16. The method of claim 15,wherein the human subject achieves a Crohn's Disease Activity Index(CDAI) score of <150.
 17. The method of claim 2, wherein the humansubject has ulcerative colitis.
 18. The method of claim 3, wherein thehuman subject has Crohn's disease.
 19. The method of claim 18, whereinthe human subject achieves a Crohn's Disease Activity Index (CDAI) scoreof <150.
 20. The method of claim 3, wherein the human subject hasulcerative colitis.
 21. The method of claim 1, wherein each subcutaneousadministration is with a prefilled syringe.
 22. The method of claim 2,wherein each subcutaneous injection is administered to the human subjectusing a prefilled syringe.
 23. The method of claim 3, wherein eachsubcutaneous injection is administered to the human subject using aprefilled syringe.
 24. The method of claim 5, wherein the human subjecthas Crohn's disease.
 25. The method of claim 24, wherein the humansubject achieves a Crohn's Disease Activity Index (CDAI) score of <150.26. The method of claim 5, wherein the human subject has ulcerativecolitis.
 27. The method of claim 6, wherein the human subject hasCrohn's disease.
 28. The method of claim 27, wherein the human subjectachieves a Crohn's Disease Activity Index (CDAI) score of <150.
 29. Themethod of claim 6, wherein the human subject has ulcerative colitis. 30.The method of claim 6, wherein each subcutaneous injection isadministered to the human subject using a prefilled syringe.